Hepatites viruses. Methods of diagnosis, treatment and prophylaxis of these diseases.

 

Retroviruses. HIV. Laboratory diagnosis of HIV infection/AIDS. Oncogenic viruses. Slow viral infections. Prions

 

Hepatitis viruses

Although hepatitis (inflammation of the liver) was first described in the fifth century BC , it is only recently (1940 to 1950) that the viral etiology of man cases of this disease has been established. More than 50,000 cases of viral hepatitis are reported annually in the United States.

Human hepatitis is caused by at least six genetically and structurally distinct viruses (Table  1). The diseases caused by each of these viruses are distinguished in part by the length of their incubation periods and the epidemiology of the infection. This chapter discusses the structure and replication of these five viruses, designated hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis h virus (HFV), and hepatitis B associated delta virus (HDV).

 

Characteristics of Human Hepatitis Viruses

 

HAV – hepatitis A virus,  HBV – hepatitis B virus,  HCV – hepatitis C virus,  HDV – hepatitis D virus,  HEV – hepatitis E virus.

* Length of incubation will vary depending on the HBV status of the infected individual. HDV infection requires  either coinfection with HBV or prior infection with HBV

 

Hepatitis A Virus

STRUCTURE AND REPLICATION OF HEPATITIS A VIRUS. Hepatitis A virus, now classified as a member of the Picornaviridae, is a spherical, R.NA containing particle, 27 to 32 nm in diameter. Biochemical analysis has shown that the virus possesses a single stranded RNA of about 7500 nucleotides. The mature virus particle contains three major polypeptides (VP1, VP2, and VP3) with molecular weights ranging from 14,000 to 33,000 daltons. The particle also can contain a small VP4 protein.

HAV is one of most stable viruses infecting humans. It is resistant to treatment with diethyl ether, can withstand heating at 56 °C for 30 minutes, and is remarkably  resistant to many disinfectants/ Electron microscopy of fecal extracts mixed with antibody to HAV has revealed clumps of virus particles about 27 nm in diameter with icosahedral symmetry. Although minor biochemical differences have been reported among HAV strains isolated in different studies, there appears to be no evidence for major  antigenic differences among HAV strains isolated in various parts or the world.

 

EPIDEMIOLOGY OF HEPATITIS A VIRUS INFECTIONS. The spread of hepatitis A is most often from person to person by a fecal-oral route, hence, the older term for the disease was infectious hepatitis. An average of 25,000cases of hepatitis A are reported each year in the United States. However, these cases represent only a small percentage or actual infections, because many HAV infections remain undiagnosed. This is particularly true for children, in whom infections frequently arc subclinical and the characteristic jaundice rarely is seen. As public health standards increase, the overall prevalence of HAV usually decreases However, especially in developing countries, this can lead paradoxically to more disease, because it often postpones exposure to the virus until an age at which infection is more likely to produce clinical symptoms.

The most common source of infection is close person to person contact. Outbreaks of hepatitis A have been reported in day-care centres and institutions for the mentally retarded. In some cities in the United States, 9% to 12% of reported cases of hepatitis A occur in children in day-care centres, their parents, or staff members. Epidemics also have resulted from drinking fecally contaminated water; however, such water borne epidemics are rare. Eating food prepared by an infected person or ingesting raw oysters, clams, or mussels harvested from fecally contaminated water is the source of many HAV infections. Because there is no persistent infection with continuous viremia (as in HBV infections), HAV transmission by blood products is rare. Although the incidence of HAV infections in intravenous drug abusers is high, it has not been proven that this is due to blood borne transmission

PATHOGENESIS OF HEPATITIS A VIRUS INFECTIONS. Hepatitis A is an acute, usually self limiting disease with an asymptomatic incubation period of 15 to 40 days. During this time, the liver is infected and large amounts of virus can be shed in the feces. Symptoms usually begin abruptly with fever, nausea, and vomiting (Table 2).The major area of cell necrosis occurs in the liver, and the resulting enlargement of the liver frequently causes blockage of the biliary excretions, resulting in jaundice, dark urine, and clay colored stool. A fulminant form of hepatitis A occurs in only 1% to 4% of patients. Complete recovery can require 8 to 12 weeks, especially in adults. During convalescence, patients frequently remain weak and occasionally mentally depressed.

In humans, the severity of the disease varies considerably with age, most cases occurring in young children are mild and undiagnosed, resolving without sequelae. In contrast to HBV, HAV infections result io extrahepatic manifestations of acute infection and no long term carrier state, and they are not associated with either cirrhosis or primary hepatocellular carcinoma.

DIAGNOSIS OF HEPATITIS A VIRUS INFECTIONS. The diagnosis of individual cases of hepatitis A usually is not possible without supporting laboratory findings. However, a tentative diagnosis of hepatitis A is appropriate if there is the simultaneous occurrence of several cases in which the epidemiology and incubation period are consistent with that of HAV disease Such situations have been known to arise in day-care centres, summer camps, and military installations.

Virus particles frequently can be detected in fecal extracts by use of immune electron microscopy, in which the fecal extract is mixed with antibodies to HAV. Standard radioimmunoassays also can be used to detect the presence of HAV antigens in fecal extracts. An enzymelinked immunosorbent assay using anti HAV linked to either horseradish peroxidase or alkaline phosphatase also is used to detect fecal HAV.

In addition, a specific diagnosis of hepatitis A can be made by demonstrating at least a four fold rise in anti-HAV antibody levels in serum.

CONTROL OF HEPATITIS A VIRUS INFECTIONS. Proper sanitation to prevent fecal contamination of water and food is the most effective way to interrupt the fecal-oral transmission of hepatitis A.

Pooled immune serum globulin from a large number of individuals can be used to treat potentially exposed poisons, and its effectiveness has been well established. Immune serum globuliormally contains a substantial titer of neutralizing antibodies to HAV. Studies indicate that large amounts of immune serum globulin can effectively prevent hepatitis A infection, whereas smaller amounts (0 01 mg/kg) modify the severity of the disease, resulting in a mild or asymptomatic infection Such infections can produce a long-lasting active immunity.

Formalin inactivated HAV vaccines have been developed and some have been licensed. Additional approaches using recombinant DNA techniques also are being used to generate subunit vaccines or novel recombinant vaccine strains

 

Hepatitis B Virus

About 300 million people world-wide are thought to be carriers of HBV, and many carriers eventually die of resultant liver disease HBV causes acute hepatitis that can vary from a mild and self limiting form to an aggressive and destructive disease leading to postnecrotic cirrhosis. Many HBV infections are asymptomatic(especially in children). However, many infections become persistent, leading to a chronic carrier state. This can lead to chronic active hepatitis and cirrhosis later in life. The HBV carrier state also is strongly associated with one of the most common visceral malignancies world-wide, primary hepatocellular carcinoma. Much of our early knowledge concerning HBV infections stems from studies with human volunteers, because the virus does not readily infect cell cultures More recently, the application of molecular biologic techniques, especially recombinant DNA technology, has yielded significant insights into the structure and replication of HBV.

 

Table

Differential Characteristics of Hepatitis A and Hepatitis B

 

STRUCTURE OF THE HEPATITIS B VIRION

In spite of our inability to grow HBV in cell cultures, several details have been learned about the structure of the hepatitis B virion through studies of new antigens appearing in the blood of infected persons Such information indicates that HBV is unlike any known group of human viruses. Interestingly, similar viruses have been identified in other species Woodchucks, Beechy ground squirrels, and Peking ducks all harbour viruses that are similar in structure and in biologic properties to human HBV.

In 1964, it was discovered that numerous virus-like particles were present in the blood of both patients with HBV hepatitis and asymptomatic carriers of HBV. These virus-like particles, first discovered in the serum of an Australian aborigine, originally were referred to as Australia antigen or hepatitis-associated antigen. The particles are uniformly 22 nm in diameter, existing as both spherical particles and filaments (Fig. 1). Treatment with ether removes a 2-nm envelope, leaving a 20-nm particle. However, these particles do not contaiucleic acid and since have been shown to represent incomplete virus particles containing HRV envelope protein but lacking nucleocapsids. The standard terminology for these particles is HBsAg to designate that they contain the surface antigens of HBV.

FIGURE.  Fraction of the blood scrum from a patient with a severe ease of hepatitis. The larger spherical particles, or Dane particles, are 42 nm in diameter and are the complete hepatitis B virus. Also evident are filaments of capsid protein (HBsAg).

 

In 1970, another particle, 42 nm in diameter, was found in the serum of patients with hepatitis B. These larger particles (named Dane particles after their discoverer) occurred in much lower concentrations than did the HBsAg particles. Dane particles were shown to contain the double-stranded, circular viral DNA genome. It has now been demonstrated that the 42-nm Dane particle represents the intact, infectious HBV particle.

 

Treatment of the Dane particles with a non-ionic detergent dissociates the HBsAg and liberates a 27-nm inner core. This inner core contains a core protein, defined serologically as the HBcAg, as well as viral DNA. It also contains two virally encoded enzymes (a DNA polymerase and a protein kinase).

Another HBV antigen, designated HBcAg, is often found in the serum of patients during the early stages of infection and in patients with chronic active hepatitis. HBcAg is structurally related to the HBV core protein and is encoded by the C gene. It can be detected in preparations of Dane particles and, therefore, appears to be an integral part of the infectious virion. Its presence in serum is believed to reflect active replication of HBV and is a marker for active disease. The appearance of anti-HBc antibodies generally correlates with a good prognosis and a decline in virus replication.

 

REPLICATION OF HEPATITIS B VIRUS. Studies on the replication of HBV and HBV-related vi-ruses (ie, woodchuck, ground squirrel, and duck hepatitisviruses) have suggested a unique mode of replication for HBV. This replication involves reverse transcription, indicating that HBV is phylogenetically related to the retrovirus family. The viral genome of HBV is about 3000 to3300 nucleotides in length, and molecular cloning and DNA sequencing experiments have established the relative organization of the genes for the various structural proteins (Fig. 2). In addition, an open reading frame encoding a putative DNA polymerase has been identified.

Although the viral DNA is circular, both strands of the duplex are linear, and the circular conformation is maintained solely by extensive base pairing between the two gapped DNA strands. Within the virus particle, the negative strand appears to be uniform in length, about 3200 nucleotides. In contrast, the positive strand is shorter and varies in length between different virions, due to single-stranded gaps of variable size. On infection, the DNA polymerase in the nucleocapsid core is activated and completes the synthesis of the positive strand, using the negative strand as a template.

The important steps of this model of HBV replication are summarized in Figure 3. After the conversion of gapped double-stranded viral DNA to fully double-stranded DNA, a full-length positive-strand RNA (a “pre-genome”) is transcribed from the HBV DNA template. This RNA serves as the mRNA for the translation of the HBcAg. Evidence suggests that this form of RNA also is packaged with viral core proteins and the viral DNA polymerase within the cell to form an “immature core”. A DNA strand of negative polarity then is synthesized through reverse transcription. This step is followed by the synthesis of a partial positive strand and the full maturation of the virus particle containing a gapped DNA genome.

 

 

FIGURE. Organization of the genes in hepatitis B virus (HBV). The dashed line represents the variable single- stranded region. The EcoRI site denotes the point of origin for the physical map. The broad arrows define the four large open reading frames of the L strand transcript. The four coding regions arc designated S(made up of pre-S and S genes), P (polymerise), X (regulatory gene), and C. The two regions encoding the S (surface antigen) and C (core antigen) proteins are represented by stippling.

 

EPIDEMIOLOGY OF HEPATITIS B VIRUS INFECTIONS. Early volunteer studies failed to show a normal portal of exit for HBV and, for years, it was believed that a person could become infected only by the injection of blood or serum from an infected person or by the use of contaminated needles or syringes. As a result, the older name for this disease was serum hepatitis. It has now been shown that this supposition is not true. Using serologic techniques, HBsAg has been found in feces, urine, saliva, vaginal secretions, semen, and breast milk. Undoubtedly, the mechanical transmission of infected blood or blood products is one of the most efficient methods of viral transmission, and infections have been traced to tattooing, ear piercing, acupuncture, and drug abuse. About5% to 10% of intravenous drug abusers are HBV carriers, and as many as 60% show evidence of previous HBV infections. Neonatal transmission also appears to occur during childbirth. The incidence is increased significantly if the mother’s blood contains HBcAg. For example, in a study from Taiwan, a 32% transmission rate was observed, and the transmission could be correlated with HBcAg-positive cord blood. The presence of HBsAg in breast milk also suggests an additional vehicle for the transmission of HBV to the newborn. The demonstration of infectious virus in semen presents the possibility that virus can be sexually transmitted. In hospitals, HBV infections are a risk for both hospital personnel and patients because of constant exposure to blood and blood products.

 

PATHOGENESIS OF HEPATITIS B VIRUS  INFECTIONS. Acute hepatitis caused by HBV cannot be clinically distinguished from hepatitis caused by HAV. However, several characteristics differentiate the infections caused by these viruses (see Table 2). HBV infections are characterized by a long incubation period, ranging from 50 to 180 days. Symptoms such as fever, rash, and arthritis begin insidiously, and the severity of the infection varies widely. Mild cases that do not result in jaundice are termed anicteric. In more severe cases, characterized by headache, mild fever, nausea, and loss of appetite, icterus (jaundice)occurs 3 to 5 days after the initial symptoms. The duration and severity of the disease vary from clinically inapparent to fatal fulminating hepatitis. The overall fatality rate is estimated to be 1% to 2%, with most deaths occurring in adults older than 30 years of age. The duration of uncomplicated hepatitis rarely is more than 8 to 10 weeks, but mild symptoms can persist for more than 1 year. The mechanism of hepatic damage of HBV is not established, but considerable data support the notion that most of the liver damage that occurs during acute or chronic hepatitis is mediated by a cellular immune response directed toward the new antigens deposited in the cell membrane of the infected cell.

Based on the ultimate pattern of the disease, this disease can be divided into two categories: self- limiting acute infections and chronic infections.

FIGURE. A model for the replication of hepatitis B like viruses. See text for details.

 

 

Self-Limiting Hepatitis B Virus Infections. Self limiting infections can be inapparent or can result in a clinical hepatitis with jaundice lasting 4 to 5 weeks. HBsAg may or may not be present in the blood, but, if present, it usually disappears as the symptoms of hepatitis subside and the jaundice clears. Antibodies to HBcAg, HBeAg, and HBsAg arise at different periods during the infection and can remain detectable for years after recovery. There seems to be a good immune response to groupspecific determinants, because recover)’ appears to provide immunity to different subtypes of the virus.

Chronic Hepatitis B Virus Infections. Between 6% and 10% of clinically diagnosed patients with hepatitis B become chronically infected and continue to have HBsAg in their blood for at least 6 months, and sometimes for life. Chronic infections can be subdivided into two general categories: chronic persistent hepatitis and chronic active hepatitis. The latter is the most severe and often eventually leads to cirrhosis or the development of primary hepatocellular carcinoma. Worldwide, it has been estimated that there are more than 200 million permanently infected carriers of HBV, of which about 1million reside in the United States. The prevalence of chronic carriers varies widely in different parts of the world, from 0.1% to 0.5% in the United States to up to 20% in China, Southeast Asia, and some African countries. The perinatal infection of newborn infants born to chronically infected mothers results in a high incidence of chronic infection (90%), which often is lifelong. This is particularly disquieting in the developing countries of Asia and Africa, where carrier rates are high. It has been estimated that HBV is the most common single cause of liver disease in the world.

All carriers have antibodies to HBcAg, and some have antibodies to HBeAg. Those who do not possess antiHBe may have circulating HBeAg. Carriers with high concentrations of Dane particles and circulating HBeAg appear to be more likely to suffer liver damage than those in whom only HBsAg can be detected, but the validity of this proposal is yet to be established. However, such persons are much more likely to be transmitters of the disease than are those who have solely HBsAg in their blood. Several cases of membranous glomeulonephritis have been described in HBsAg-positive children, and it has been reported that the glomerulonephritis results from the deposition of immune complexes consisting of anti-HBe IgG and HBeAg.

The mechanism by which carriers can remain persistently infected and yet be asymptomatic is unknown. However, prolonged carrier status is seen in association with chronic hepatitis in patients with lowered immunity and in those infected during the neonatal period or early childhood.

Virus-Host Immune Reactions. Currently there is evidence for at least 3 hepatitis viruses—type A (short incubation hepatitis virus), type B (long incubation hepatitis virus), and the agent or agents of non-A, non-B hepatitis. A single infection with any confers homologous but not heterologous protection against reinfection. Infection with HBV of a specific subtype, eg, HBsAg/adw, appears to confer immunity to other HBsAg subtypes, probably because of their common group a specificity.

Most cases of hepatitis type A presumably occur without jaundice during childhood, and by late adult­hood there is a widespread resistance to reinfection. However, serologic studies in this country indicate that the incidence of infection among certain populations may be declining as a result of improvements in sanita­tion commensurate with arise in the standard of living. It has been estimated that as many as 50-75% of young middle to upper income adults in the USA may be susceptible to type A hepatitis. Younger people who live in poorer circumstances or crowded institutions (eg, the armed forces) are at increased risk.

The immunopathogenetic mechanisms that result in viral persistence and hepatocellular injury in type B hepatitis remain to be elucidated. An imbalance be­tween suppressive and cytopathic immune responses of the host has been hypothesized to account for the various pathologic manifestations of this disease. It is postulated that antibody-dependent, complement-mediated cytolysis or cellular effector mechanisms are responsible for the hepatic injury observed, whereas noncytopathic synthesis of viral components, surface expression of viral antigens or liver-specific neoantigens, and shedding of virus are primarily modulated by the humoral immune response.

Various host responses, immunologic and ge­netic, have been proposed to account for the higher frequency of HBsAg persistence observed in infants or children compared to adults and in certain disease states, eg, Down’s syndrome, leukemia (acute and chronic lymphocytic), leprosy, thalassemia, and chronic renal insufficiency. Patients with Down’s syn­drome are particularly prone to persistent antigenemia (but low antibody frequency) and inapparent infec­tions, and they show a significantly greater prevalence of these disorders than is found in other mentally retarded patients. This does not imply that these pa­tients have an increased susceptibility to HBV. On the contrary, among other equally exposed patients who are residents within the same institution, the total serologic evidence of HBV infection is similar except that the antigen carrier rate is low whereas the antibody prevalence is high. An immunologic difference in the host response to the virus is apparently responsible for this serologic dichotomy.

Persistent antigenemia and mild or subclinical infections are more frequently observed in individuals who have been infected with low doses of virus. Corre­spondingly, a direct relationship between virus dose and time of appearance of HBsAg or an abnormal ALT value has been reported; i.e., the incubation period be­comes longer as the dose of virus diminishes.

The frequency of the chronic HBsAg carrier state following acute icteric type B hepatitis is not known but is probably under 10%. More than half of these patients continue to exhibit biochemical and histologic evidence of chronic liver disease, i.e., chronic persistent or chronic active hepatitis.

Primary Hepatocellular Carcinoma. A considerable amount of evidence has documented the close association between HBV infection and the development of primary hepatocellular carcinoma. Hepatocellular carcinoma is the most common cancer in the world, with at least 250,000 new cases reported annually. Patients with hepatocellular carcinoma often have high levels of HBsAg, and the carcinoma cells often contain integrated HBV DNA. Further evidence for the link between persistent HBV infections and hepatocellular carcinoma comes from epidemiologic data showing that the risk of developing primary hepatocellular carcinoma is more than 200 times higher in HBV carriers than ioncarriers. Within some populations, the risk of developing primary hepatocellular carcinoma is as high as 50% in male chronic carriers. However, HBV infection is not solely responsible for tumor development, because the carrier state often exists for a lengthy period (often 40 years or more) before the onset of liver cancer In addition, the predominance of hepatocellular carcinoma in men indicates that other factors, including sex related factors, contribute to the development of this cancer. Nonetheless, an important component of chronic liver disease is the continual regeneration of damaged or destroyed hepatocytes, which, coupled with HBV replication and exposure to environmental carcinogens, likely contributes in a significant fashion to tumor development and progression. The relationships between oncogene activation, loss of tumor suppressor genes, and the HBV are under active investigation.

Because of the close egidemiologic link between chronic HBV infection and hepatocellular carcinoma, it is hoped that mass vaccination of susceptible individuals in such countries as China and Taiwan will reduce the overall incidence of HBV infection, and that this eventually will reduce dramatically the incidence of hepatocarcinoma.

DIAGNOSIS OF HEPATITIS B VIRUS INFECTIONS. As in all cases of viral hepatitis, abnormal liver function is indicated by increased levels of liver enzymes such as serum glutamic oxaloacctic transaminase and alanine aminotransferase (ALT). The presence of HBsAg confirms a diagnosis of hepatitis B, and its serologic detection is routinely carried out in diagnostic laboratories and blood banks using radioimmunoassays or enzyme-linked immunosorbent assay’s.

 

 

CONTROL OF HEPATITIS B VIRUS INFECTIONS. The examination of all donor blood for the presence of HBsAg is now routine, and this practice has done much to control the occurrence of posttransfusion hepatitis B infections.

Passive immunization of human volunteers with hepatitis B immune globulin (HBIG) has been shown to prevent disease when the volunteers were challenged with infectious material, but the use of immune globulin is not effective for the treatment of active disease. One important and effective use for HBIG, however, is the prevention of active hepatitis B infections ieonates born to mothers who are chronic carriers of HBsAg. HBIG also can be given to nonimmune individuals known to have been exposed to HBV.

Active immunization with HBsAg promises to provide a vehicle for the control of hepatitis B. Clinical trials in high-risk populations have shown that the incidence of hepatitis B in persons actively immunized with HBsAg is decreased by about 95%. Moreover, immunization even during the long incubation period may be efficacious in preventing HBV infections. Because HBV has not been grown in cell cultures, the first vaccine consisted of highly purified, formalin-inactivated HBsAg particles obtained from the plasma of persistently infected carriers. This vaccine has now been superseded by a recombinant vaccine, in which the gene for HBsAg has been cloned in yeast, enabling the production of polypeptides carrying the antigenic determinants of HBsAg in large amounts. The yeast-produced vaccine has been licensed for use and has been given to more than 2 million people in the United States. The vaccine is considered safe and provides effective protection. Administration of the HBV vaccine world-wide has the potential to reduce drastically the incidence of HBV infection. Early studies have shown that its use in HBV-positive pregnant women reduces the percentage of infants who become carriers from 90% to 23%. In addition, if HBIG is used in conjunction with the vaccine, the newborn carrier incidence can be reduced to less than 5%. Taking note of the fact that many chronic HBV carriers eventually die of liver disease, tills vaccine represents the first prophylactic measure to substantially reduce or prevent cirrhosis and human cancer.

Non-A, Non-B Hepatitis. About 20 years ago, as diagnostic assays to detect HAV and HBV became readily available, it was demonstrated that most cases of transfusion-associated infection were caused by neither HAV nor HBV. Thus, it seemed clear that other hepatitis viruses remained to be isolated. The disease caused by these unknown agents became known as non-A, non-B (NANB) hepatitis. It has now been shown that most cases of transfusion-associated hepatitis are caused by an RNA virus that has beeamed HCV. Two other RNA viruses responsible for some cases of NANB hepatitis also have been identified. One of these viruses (HDV) requires HBV to replicate and, therefore, is seen only in individuals who are infected with HBV. A third RNA agent of NANB hepatitis, which is called HEV and is spread by a fecal-oral route, has been shown to be the cause of large outbreaks of hepatitis in developing countries.

HEPATITIS C VIRUS. When it became clear that most cases of transfusion-associated hepatitis probably were caused by a hitherto unknown virus, molecular genetic and recombinant DNA techniques were used to identify, clone, and sequence putative agents. This led to the isolation of a new RNA virus, HCV. Sequence analysis has revealed that HCV is organized in a manner similar to the flaviviruses and that it shares biologic characteristics with this family. This has led to a classification of HCV as a genus within the flavivirus family. About 80% of patients with chronic, post-transfusion NANB hepatitis in Italy and Japan have been shown to have antibodies to HCV, and 58% of patients with NANB hepatitis in the United States, with no known parenteral exposure to the virus, have HCV antibodies. Based on these data, it seems likely that HCV is a major contributor to NANB hepatitis throughout the world. Most infected individuals become chronic carriers of the virus, and many develop chronic hepatitis. Studies in several urban areas have shown that as many as 80% of intravenous drug abusers have been infected with HCV. The development of commercial antibody tests to detect HCV infection has markedly reduced the number of cases of NANB hepatitis acquired from transfusions and blood products.

 

 

HEPATITIS DELTA VIRUS. Hepatitis delta virus was first described in 1977 as a novel antigen-antibody complex detected by immunofluorescence in hepatocyte nuclei of patients with chronic HBV infection and chronic hepatitis. Although HDV antigen was initially observed in Italy, it has been detected world-wide, primarily in HBV carriers who have had multiple exposures to blood and blood

Table

Prevalence of Delta Infection in Hepatitis B Virus (HBV) Carriers and

Persons With HBsAg-Positive Acute and Chronic Hepatitis in North Americae

 

Transmission experiments in chimpanzees and other studies have shown that HDV is a transmissible and pathogenic agent that requires concomitant replication of HBV to provide certain helper functions. The HDV virion is a spherical, 36-nm enveloped particle with a chimeric structure; the genome consists of a 1.7-kilobase RNA molecule specific for HDV, whereas the envelope contains HBV encoded HBsAg. The HDV genomic RNA is a circular, single stranded RNA similar in structure to certain pathogenic RNAs or plants (viroids), and its replication requires the concomitant expression of HBV gene products

Two principal modes of HDV infection have been described (1) coinfection (the simultaneous introduction of both HBV and HDV into a susceptible host), and(2) superinfection (the infection of an HBV carrier with HDV). Simultaneous exposure to HBV and HDV leads to a typical pattern of HBV disease, with the duration of HBV infection being the limiting factor to the expression of HDV. The outcome of such HBV/HDV coinfections usually is similar to that of infection with HBV alone, and chronic infections seem to be established with the same frequency.

The clinical outcome from HDV superinfection of an HBV carrier is markedly different In this case, the persistent HBV infection promotes the efficient replication of the defective HDV and leads to a fulminant HBsAg-positive hepatitis with a significant mortality rate (5% to 15%). In addition, the chronic infection with HBV potentiates the continued replication of HDV, establishing a chronic HDV infection. There are few data to support a role for HDV in the development of primary hepatocellular carcinoma.

HDV transmission is linked closely to that of its helper, HBV. Parenteral inoculation accounts for the world-wide distribution of HDV among drug addicts. In parts of the world with a low incidence of HBV, HDV infections are found mostly in drug addicts and other individuals at risk for being HBV carriers HDV infection of newborns occurs only in babies born to HBcAg-positive, HDV infected mothers. Although HDV is found worldwide, an interesting anomaly exists in that HDV infection is endemic in South America, resulting in severe outbreaks of fulminant hepatitis. In contrast, HDV infections are rare in Asia, although the prevalence of HBsAg carriers is similar to that in South America. Overall, it has been estimated that about 5% of chronic HBV carriers also are infected with HDV.

Because no HDV vaccine is available, controlling the transmission of HBV is the only approach to controlling the spread of HDV. Unfortunately for the estimated 200 million HBsAg carriers in the world, there is no effective measure to prevent HDV infection per se.

 

HEPATITIS E VIRUS. Many cases of acute viral hepatitis in Asia and Africa are caused by a virus that is transmitted through the fecal-oral route but is unrelated to HAV. Outbreaks of this disease also have been confirmed in other parts of the world, including the Middle East and Mexico. The disease usually is caused by the ingestion of fecally contaminated water. The virus causing this kind of hepatitis has beeamed HEV. The first verified hepatitis E outbreak was documented in New Delhi, India, in 1955 In this epidemic, 29,000 cases of icteric hepatitis were reported after fecal contamination of the city’s drinking water Several other outbreaks have been linked to HEV since then HEV is a small, nonenveloped RNA virus. Recent information about the genomic organization and other properties of the virus strongly suggests that it is a calicivirus and should be placed in a new genus within this family.

 

Additional material about diagnosis of hepatites

HEPATITIS A

The hepatitis A virus belongs to the family Picornaviruses, genus Enterovirus, type 72,

The patient’s faeces should be collected for examination. To iso­late the virus, a 10-40 per cent faecal extract homogenated in phos­phate buffer (pH 7.4) is prepared. Gross particles are removed by slow velocity centrifugation. The virus is concentrated using differ­ential centrifugation combined with extraction by organic sol­vents (chloroform), filtration through agarose (sefarose CL-2B), and density centrifugation in caesium chloride. The highest concentra­tions of the virus in patients’ faeces, reaching 10⁶ and more virions per g of faeces, are noted several days before the onset of clinical manifestations of the infection (at the end of the incubation period). With the onset of a manifest infection the faecal concentration of the virus progressively decreases.

Rapid diagnosis is based on IEM, RIA, and ELISA.

Viral particles in the faecal extract can be detected by IEM only when their concentration is at least 10⁴. The faecal extract (10-20 per cent) is mixed with a specific serum in a 9:1 ratio and incubat­ed at 37 °C. Sedimentation is performed by centrifugation at 10 000 X g for 30 min; the residue is examined under the electron microscope.

Solid phase RIA consists of three stages: (a) adsorption of antibod­ies on the polyvinyl surface of test tubes; (b) binding of the antigen from the faecal extract by fixed antibodies; (c) demonstration of the adsorbed antigen by specific antibodies labelled with radioactive io­dine. The preparation of labelled antibodies should contain 1-2 atoms of radioactive iodine per molecule of gamma-globulin.

ELISA, which allows demonstration of the viral antigen in the faecal extract with the help of a specific serum and an enzyme-linked antiserum, presents a highly sensitive test.

Isolation and identification of the virus is based on inoculating sensitive animals (chimpanzee and marmoset monkeys) as well as cultures of human lymphocytes stimulated with phytohaemagglutinin with the filtrate of faeces. The viral antigen is determined by means of the IF reaction in the cytoplasm of hepatocytes. Electron microscopy is useful in detecting aggregates of the viruses.

Serological examination is based on the demonstration of specific IgM which appear very early, simultaneously with the rise in serum enzymes and IgG. To detect antibodies of the IgM and IgG classes, IEM, CF, RIA, and ELISA are employed. Preparations of the purified and concentrated virus isolated from patients’ faeces are used as an antigen.

 

HEPATITIS B

The hepatitis B virus and three analogous viruses affecting ani­mals are referred to the family Hepadnaviridae, the hepatitis B being denoted as type 1 hepatitis virus.

The virus contains three antigens, surface HBsAg and two inter­nal ones: HBcAg (median) and HBeAg. The latter exhibits the properties of DNA-polymerase. At different stages of the disease, the patient’s body forms antibodies (anti-HBs, anti-HBc, and anti-HBe) to each of the antigens.

Rapid diagnosis. In acute viral hepatitis HBsAg can usually be demonstrated in patients’ serum in the incubation period, namely, 2-8 weeks prior to biochemical changes and elevation in the activity of aminotransferases. It should be noted that HBsAg can be detected in only 50-80 per cent of patients, which means that a negative re­sult does not rule out the possibility of virus hepatitis B.

There are different methods of recovering HBsAg in the blood serum (ELISA, RIA, RIHA, precipitation in gel). Counterimmunoelectrophoresis can also be used for this purpose. To enhance the specificity of these reactions, it is recommended that sera be concentrated by drying them in a 37 °C incubator and subsequent dilution in a smaller volume of distilled water.

Serological examination. To detect antibodies to the antigens of hepatitis B virus, such tests as precipitation in gel and counter-immunoelectrophoresis are utilized. The most sensitive and specific are RIA, ELISA, and I HA with the use of HBsAg-loaded red blood cells.

Determination of the antigens of hepatitis B virus and antibodies to them is important not only for the diagnosis of virus B hepatitis but also for predicting its outcomes, which is explained by the fact that different stages of the disease are associated with different markers of the hepatitis B virus. The incubation period is character­ized by the presence in the blood of HBsAg which usually persists for 2-5 months, retaining, however, much longer in the blood in chronic cases of the disease. In the acute period of the disease, HBeAg and HBeAg make their appearance. The latter can circulate in the blood serum for 1-7 weeks, its presence for 3 weeks from the onset of the disease being prognostically unfavourable. A stage of early convalescence is characterized by the disappearance from the blood serum of HBcAg and HBeAg and the appearance in it of anti-HBc (in increasing titres); anti-HBe may also be found. At a stage of late convalescence, antibodies to all three antigens of hepatitis B virus are demonstrated in the blood serum.

Chronic aggressive hepatitis B is characterized by the appearance in the blood of HBsAg and HBeAg and also by high titres of anti-HBc IgM, which evidences continuing replication of the virus.

In carriers of HBsAg, the examination of the serum reveals, in addition to this antigen, low titres of antibodies (anti-HBc, anti-HBe IgM, and anti-HBc IgG; in rare cases anti-HBs may be ob­served).

This form of hepatitis is not uncommonly associated, apart from Dein’s particles, with another type of viral particles, namely, delta-particles (or delta-antigen). A distinctive feature of delta-particles is the dependence of their reproduction on the reproduction of Dein’s particles. These are small RNA-containing viruses whose surface (capsid) protein is represented by HBsAg.

 

Laboratory Features. Liver biopsy permits a tissue diagnosis of hepatitis. Tests for abnormal liver function, such as serum alanine aminotransterase (ALT; formerly SGPT) and bilirubin. supplement the clinical, pathologic, and epidemiological findings. Transaminase val­ues in acute hepatitis range between 500 and 2000 units and are almost never below 100 units. ALT values are usually higher than serum aspartate transaminase (AST; formerly SCOT). A sharp rise in ALT with a short duration (3-19 days) is more indicative of viral hepatitis A, whereas a gradual rise with prolongation (35-200 days) appears to characterize viral hepatitis B and non-A. non-B infections.

Leukopenia is typical in the preicteric phase and may be followed by a relative lymphocytosis. Large atypical lymphocytes such as are found in infectious mononucleosis may occasionally be seen but do not exceed 10% of the total lymphocyte population.

Further evidence of liver dysfunction and host response is reflected in a decreased serum albumin and increased serum globulin. Elevation of gamma globu­lin and serum transaminase is frequently used to gauge chronicity and activity of liver disease. In many pa­tients with hepatitis A, an abnormally high level of IgM is found that appears 3-4 days after the ALT begins to rise. Hepatitis B patients have normal to slightly elevated IgM levels.

The most sensitive and specific method for detect­ing HBsAg or anti-HBs is the radioimmunoassay (RIA). This test and the red cell agglutination (RCA) technique, which employs HBs antibody-coated cells in a microtiter system, have replaced counterelectrophoresis as the methods of choice for detecting HBsAg. The passive hemagglutination (PHA) technique, which uses HBs antigen-coated cells, is an excellent and rapid method for detecting anti-HBs, rivaling RIA in sensitivity. The enzyme-linked immunosorbent assay (ELISA) has recently gained acceptance in many countries be­sides the USA because it circumvents the relatively short half-life of isotopes inherent in RIA systems.

The particles containing HBsAg are antigenically complex. Each contains a group-specific antigen, a, in addition to 2 pairs of mutually exclusive subdeterminants, dly and wir. Thus, 4 phenotypes of HBsAg have been observed: adw, ayw, adr, and ayr. In the USA, adw is the predominant subtype among asymptomatic carriers, whereas ayw has frequently been observed in dialysis-associated outbreaks and among parenteral drug abusers. These virus-specific markers are useful in epidemiological investigations, since sec­ondary cases have the same subtype as the index case. The evidence indicates that these antigenic determi­nants are the phenotypic expression of HBV genotypes and are not determined by host factors.

The clinical and serologic events following expo­sure to HBV are depicted in Fig. 4 and in Table 5. DNA polymerase activity, which is probably representative of the viremic stage of hepatitis B, oc­curs early in the incubation period, coinciding with the first appearance of HBsAg.

Table

Common serologic tests for HBV and their interpretation

 

*0ther HBV serologic markers that may be present at the same time include Dane particles (HBV), observable by electron microscopy. Core antigen and viral DNA polymerase can be measured by disrupting HBV.

 

Figure. Clinical and serologic events occurring in a patient with hepatitis type B.

 

The latter is usually de­tectable 2-6 weeks in advance of clinical and biochem­ical evidence of hepatitis and persists throughout the clinical course of the disease but typically disappears by the sixth month after exposure. Occasionally, HBsAg persists in patients who develop chronic active hepatitis. In patients destined to become carriers, the initial illness may be mild or inapparent, manifested only by an elevated transaminase determination.

Anti-HBc is frequently detected at the onset of clinical illness approximately 2-4 weeks after HBsAg reactivity appears. Because this antibody is directed against the internal component of the hepatitis B virion, its appearance in the serum is indicative of viral replication. In the typical case of acute type B hepatitis, the anti-HBc titer falls after recovery. In contrast, high titers of anti-HBc persist in the sera of most chronic HBsAg carriers. Antibody to HBsAg is first detected at a variable period after the disappearance of HBsAg. It is present in low concen­trations usually detectable only by the most sensitive methods.

The anti-HBc test is of limited clinical value when the HBsAg test is positive. However, in perhaps 5% of the acute cases of hepatitis B, and more frequently during early convalescence, HBsAg may be undetectable in the serum. Examination of these sera for anti-HBc may help in establishing the correct diagnosis. In the absence of anti-HBc and HBsAg, active hepatitis B disease can be excluded. In contrast, the presence of anti-HBc alone is presumptive evidence for an active HBV infection. However, this relationship is not in­fallible, and some patients who have recovered from hepatitis B with the development of anti-HBs and anti-HBc eventually lose one or the other antibody.

Another antigen-antibody system of importance involves HBeAg and its antibody. If the specimen contains HBsAg, certain situations may warrant fur­ther testing of the serum for HBeAg or anti-HBe. These include assessing the risk of transmission of HBV following exposure to contaminated blood and advising health care professionals who are chronically infected. Specimens positive for HBeAg (or positive for HBsAg at a dilution of 1:10,000) are considered to be very infectious, i.e., they contain high concentrations of HBV. Infectivity is reduced, but probably not eliminated, in specimens containing anti-HBe (or low titers of HBsAg).

The clinical, virologic, and serologic events fol­lowing exposure to HAV are shown in Fig. 5. Virus particles have been detected by immune electron microscopy in fecal extracts of hepatitis A patients. Virus appears early in the disease and disap­pears within 3 weeks following the onset of jaundice.

By means of RIA, the HAV antigen has been detected in liver, stool, bile, and blood of naturally infected humans and experimentally infected chim­panzees or marmosets. The detection of HAV in the blood of infected chimpanzees supports previous epidemiological evidence of viremia during the acute stage of the disease. Peak titers of HAV are detected in the stool about 1 -2 weeks prior to the first detectable liver enzyme abnormalities.

Anti-HAV appears in the IgM fraction during the acute phase, peaking about 3 weeks after elevation of liver enzymes. During convalescence, anti-HAV is in the IgG fraction, where it persists for decades. The methods of choice for measuring HAV antibodies are RIA, ELISA, and immune adherence hemagglutination.

Figure. Immunologic and biologic events associated with viral hepatitis type A.

 

Attempts to isolate HBV in a cell or organ culture system have generally not been successful. In contrast, HAV has recently been propagated in cell culture. Chimpanzees and some species of marmosets have been found to be susceptible to human viral hepatitis type A. HAV infections among imported chimpanzees are well known as an important cause of hepatitis in animal caretakers.

Successful transmission of HBV to chimpanzees has been achieved. The infection results in serologic, biochemical, and histologic evidence of type B hepatitis. Immunofluorescence and electron micros­copy reveal HBsAg in the cytoplasm and viruslike particles with HBcAg in the nuclei of hepatocytes. Serial passage has been successful. No evidence for hepatitis B transmission from chimpanzees to humans has been reported.

 

Retroviruses. HIV. Laboratory diagnosis of HIV infection.

Oncogenic viruses. Slow viral infections. Prions.

 

Human Retroviruses

The retroviruses are a large group of RNA viruses,  many of which readily induce neoplastic disease in their natural host.  The first of these viruses was described in the early 1900s when it was shown that leukemias and sarcomas of chickens could be transmitted to new-born healthy chickens using cell-free extracts of the tumours.  At that time,  the phenomenon was considered an intellectual curiosity,  and few scientists realized the implications of this discovery in relation to the role of viruses in  cancer.

It is now known that retroviruses are widespread iature These viruses have been isolated from a variety of vertebrate species,  including birds,  mice,  rats,  cats,  hamsters,  cattle,  horses,  and,  ecently,  humans.  Many of these RNA containing viruses cause leukemia (a malignancy of primitive blood cells such as lymphoblasts,  myeloblasts,  or erythroblasts) carcinomas,  or sarcomas (solid tumours) Several other members of the retrovirus family have been shown to cause severe immunodeficiency or neurologic disease in their respective hosts

Structure of Retroviruses. Retroviruses generally are spherical,  with an overall diameter varying from 65 to 150 nm.  The mature virion has three morphologic components: (1) an outer envelope made up of a lipid bilayer membrane containing virus specific glycoprotein spikes,  (2) an internal protein capsid; and (3) within the capsid,  a nucleocapsid and two virally encoded enzymes (reverse transcriptase and integrase). 

 

FIGURE.  Structure of the retrovirus particle.

 

The retrovirus genome is a dimeric structure consisting of two identical single stranded RNA segments (7 to 10 kilobases in length).  In addition,  unique cellular tRNAs are associated with each segment of the viral genome These tRNAs play an essential role in viral replication by providing a primer for the RNA dependent DNA polymerase (reverse transcriptase) that replicates the viral genome.

 

The retrovirus family is divided into three subfamilies: Oncovirinae,  Lentivirinae,  and Spumavirinae.  The Oncovirinae,  or RNA tumour virus subfamily,  includes all the transforming retroviruses.  Electron micrographs of different types of RNA tumour viruses reveal several morphologic types of particles.  The most common is a spherical,  enveloped virus with a centrally located dense core.  This structure has been designated as a C-type virion (Fig. A),  and it is differentiated from morphologically similar B type virions (Fig.  ),  whose spherical,  dense nucleocapsid is located eccentrically within the particle.  A type particles are observed only within the cytoplasm of a host cell and do not possess an envelope.  A-type particles,  which are often seen within the cisternae of the endoplasmic reticulum,  may represent defective retroviruses.  A fourth type of retrovirus,  designated D-type virus,  has been isolated from several nonhuman primates.  These viruses resemble B-type particles in that they have an eccentric core.  However,  they differ in structure,  having less prominent surface glycoprotein spikes.

FIGURE. A, B.            A.  Typical C-type particles of Rous sarcoma virus.  The central,  dense nucleoids are characteristic of all C-type particles.  (Original magnification X 147, 000. )

 

                                                           A                                          B                                                      

B. B-type particles of mouse mammary tumour virus in an intercellular lumen.  Note the eccentric nucleoids within the particles. (Original magnification X20, 800. )

 

The Lentivirinae subfamily (to which human immunodeficiency virus [HIV] belongs) is characterized by a distinctive nucleocapsid core,  viewed in the electron microscope as a bar or truncated,  cone shaped nucleocapsid.

The spumaviruses (from the Creek spuma,  meaning foam) derive their name from the foam like vacuoles they cause in infected cells.

 

  Replication of Retroviruses. Retrovirus replication begins with the interaction of the viral envelope glycoprotein spikes with cellular receptor proteins (if the host-cell membrane.  The cellular receptors are different for different retroviruses.  The interaction between the viral glycoprotein and the receptor activates a hydrophobic fusion protein that is part of the viral spike.  After fusion of the viral envelope with the host-cell membrane,  the viral core is released into the cell.  At this point,  the replication of the retroviruses differs from that of all other viruses,  because the first step is the copying of the viral RNA into complementary double-stranded DNA.  This reaction is catalyzed by a virus encoded RNA-dependent DNA polymerase that is present in the virus particle.  This enzyme is commonly called reverse transcriptase,  because the “transcription” it catalyzes is opposite to the normal pathway of information flow (DNA to RNA).

Viral DNA synthesis occurs initially within the cytoplasm of the host cell.  A model for viral DNA synthesis consistent with experimental data is shown in Figure 3.

DNA synthesis is initiated on a cellular tRNA primer that base pairs with the viral RNA 100 to 300 nucleotides from the 5′ end of the RNA at a location known as the primer binding site.  Synthesis proceeds in a 5′-to-3′ direction,  stopping after traversing a short sequence designated R (repeated).  The R sequence is present at both ends of the retroviral RNA genome; hence,  the newly synthesized DNA can base pair either with the sequence present at the 5′ end or with the R sequence present at the 3′ end of the RNA genome. 

 

 

 

FIGURE. Schematic diagram of retroviral DNA synthesis A,  B,  C,  D,  and E denote viral RNA sequences important for reverse transcription and arc not drawn to scale.  The sequence A represents the terminally repeated sequence R,  the sequences Band E represent the U₅ and U₃ regions of the viral RNA,  respectively; and the sequence C’ denotes the primer binding site.

 

 

 

This terminal redundancy allows a first “jump” to take place,  in which the newly synthesized DNA “jumps” to base pair with RNA sequences at the 3′ end of either the same RNA molecule (an intrastrand jump) or the second RNA molecule in the core).  After the jump has taken place,  DNA synthesis can continue,  resulting in the formation of a minus-strand DNA molecule. 

 

 

Plus-strand DNA synthesis then is initiated at a unique site in the 3′ end of the RNA and proceeds from 5′ to 3′ using the newly synthesized minus-strand DNA as a template.  The process of plus-strand synthesis again generates intermediates with complementary terminal sequences,  enabling a second jump to take place.  A complete double-stranded DNA molecule then can be made,  probably through a circular intermediate similar to that depicted in previous Figure.  The final DNA product has a longer terminally repeated sequence than the original RNA molecule because,  during the process of reverse transcription,  some sequences are copied twice.  This long terminal repeat (LTR) is made up of U₃ (a unique 3’sequence in the RNA),  R,  and Us (a unique 5′ sequence in the RNA) sequences.  The LTR sequences vary in length between different retroviruses (300 to 600 base pairs).

Linear duplex DNA synthesis takes place in the cytoplasm of infected cells,  generally within the first 8 to 24hours of infection.  At later periods,  circular forms of viral DNA are observed in the nucleus.  These DNAs contain either one or two copies of the LTR sequence.  These DNA molecules integrate into the cellular chromosomes with the help of the viral enzyme integrase.  The integration of retroviral DNA shows striking similarities with the insertion of bacterial transposable elements.  Integrated proviral DNA is identical to the linear DNA precursor except that it always lacks two base pairs present at each end of the linear precursor.  In addition,  a four-base pair repeat is generated in the host-cell target sequence.  The process of viral DNA integration requires the presence of specific sequences near the ends of the viral LTR.  sequences; these sequences are considered analogues of bacteriophage attachment (att) sites.

Integration of viral DNA generally occurs at a single site in the host chromosome,  but the site of integration varies from cell to cell.  After integration,  transcription of viral DNA is controlled by viral transcriptional elements(promoter and enhancer sequences) located within the Us sequence of the LTR (Fig. 4).  Synthesis of viral RNA,  carried out by the host cell’s RNA polymerase II,  starts at the 5′ end of R in the 5′ LTR and terminates at the 3′ end of R in the 3′ LTR.  The primary RNA product is polyadenylated using the cellular machinery.  This “full-length” RNA is used to translate the viral gag and pol proteins (see later) and also is the viral genome that gets packaged into new virus particles.  Some of the viral RNA molecules are spliced to generate smaller subgenomic mRNAs that arc used to translate env proteins(and regulatory proteins in the case of complex retroviruses such as HIV).  After processing,  the viral mRNA molecules are transported to the cytoplasm and viral proteins are synthesized on cytoplasmic polyribosomes. 

The env proteins are modified by glycosylation in the Golgi apparatus of the host cells and then are incorporated into the host-cell plasma membrane.  The assembly of viral RNA and viral gag and gag/pol proteins occurs at the cell membrane in regions where the env proteins have been incorporated,  after which the virus is released by budding from the membrane.  Unlike most other viral infections,  infections with many retroviruses do not kill the host cell,  and both host cell division and virus production can continue indefinitely.  Figure summarizes these steps in retrovirus replication.

 

 

FIGURE. Summary of the steps required for retrovirus replication.

 

RETROVIRUS GENE STRUCTURE. Many of the oncoviruses are defective (i. e. ,  they lack one or more genes necessary for replication).  These viruses can replicate only in the presence of a second helper virus.

 

Oncoviruses that can replicate independently within a host cell are termed nondefective.

Based on the biologic properties of an oncovirus in cell culture,  and on the pathology of the disease induced in an animal,  the oncoviruses can be subdivided further into two classes: acute transforming viruses and nonacute transforming viruses.  The former induce a rapid formation of leukemia or sarcoma in the host,  usually within a period of weeks,  and cause a malignant transformation of cells in culture.  Viruses belonging to the latter class cause disease in the host only after a long latent period of several months (or even years) and do nor induce malignant transformation of cells in culture,  although they replicate in such cells.  Biochemical and genetic analysis of these two classes of retroviruses has shown that the nonacute viruses contain only the genes required for viral replication: the gag gene,  encoding the structural proteins of the viral capsid and core; the pol gene,  encoding the virion-associated RNA-dependent DNA polymerase and integrase; and the env gene,  encoding the env glycoproteins of the virus (Fig.).  The nonacute viruses contain all the information required for efficient virus replication and,  therefore,  are nondefective viruses.

 

FIGURE. Organization of the genomes of various transforming and nontransforming retroviruses.  The three viral genes required for virus replication are denoted by the terms gag,  pol,  and env (see text).  The sequences acquired by transforming retroviruses by recombination with cellular sequences (here,  denoted proto-onc) are indicated by the term onc.  Insertion of one sequences into the viral genome results in the partial or complete deletion of gag,  pol,  and env sequences.  Such viruses are replication defective Rous sarcoma virus contains the onc gene denoted src inserted 3′ to the env gene and,  therefore,  is replication competent.

 

Acute transforming viruses,  on the other hand,  lack all or parts of the genes required for replication and in their place have acquired unique cellular sequences termed oncogene sequences.  It is the virus-mediated expression of these oncogene sequences that gives rise to the rapid onset of malignant disease in the animal host and to the transformation of cells in culture.

However,  because the oncogene sequences have replaced (either totally or in part) the genes required for virus replication,  acute transforming viruses are “defective” for replication and,  therefore,  require the concomitant replication of a helper virus,  a nonacute virus,  to provide the viral proteins necessary for virus replication and integration.  The one exception to this rule is Rous sarcoma virus(RSV),  an avian retrovirus.  Interestingly,  in RSV,  the resident oncogene sequence (src) lies outside the boundaries of the genes required for replication (Fig. 6). Hence,  RSV is the only known example of a nondefective, acute sarcoma virus.  For this reason,  RSV has been one of the most intensely studied retroviruses.

The lentiviruses and spumaviruses show a more complex gene structure and are characterized by the presence in the genome of one or more regulatory genes in addition to gag,  pol,  and env.  These genes play important roles in the replication of these viruses (see later).

SYNTHESIS OF RETROVIRUS PROTEINS. The retrovirus core is made up of the genomic RNA and several nucleocapsid proteins.  The multiple core proteins (known as gag [group antigen] proteins) are encoded in a single gene,  the gag gene.  They are synthesized as a polyprotein precursor,  termed Prgag,  that subsequently is cleaved into the component gag proteins by a protease usually encoded within gag.  In the case of some viruses,  the protease is encoded within pol.  A standardized nomenclature for the gag proteins that are common to all retroviruses has been established.  The individual proteins have been designated capsid protein,  matrix protein,  nucleocapsid protein,  and proteaseThe products of the pol gene,  reverse transcriptase and integrase,  also are cleaved from a precursor protein,  which is known as Prgag/pol because it contains the gag as well as the pol protein sequences.  This precursor protein is cleaved by the viral protease during the assembly process to yield the different gag proteins as well as the two pol gene products.

All retroviruses, including those of human origin, share a similar organization of genetic information. A novel and interesting feature of retrovirus translation is that gag and pol genes always are expressed as gag–pol fusion proteins, in spite of the fact that these genes are either interrupted by translational stop codons or present in different translational reading frames. This paradox is explained by the ability of eucaryotic ribosomes to occasionally insert amino acids in response to nonsense codons or, alternatively, to shift reading frames at defined sites and frequencies when translating viral RNAs.

Such mechanisms provide clear advantages to the retroviruses: structural gag proteins can be made in large amounts, whereas catalytic proteins (pol and int) are made in relatively small amounts. In addition, this process allows pol proteins to be packaged into virions attached to their gag counterparts.

The envelope gene encodes two glycoproteins that make up the envelope spikes: a larger protein forming the “knob” of the spike (also referred to as SU, surface protein), and a smaller protein forming the base (also referred to as TM, transmembrane protein). These two proteins are like the other viral gene products synthesized as a precursor protein.  They then are cleaved specifically by a cellular protease to yield the SU and TM proteins.  The surface protein binds to the receptor on the host cell,  whereas the transmembrane protein serves as a fusion protein that enables fusion of the viral and host cell membranes once binding has occurred (see earlier).

 

Exogenous Retroviruses

DEFECTIVE ACUTE TRANSFORMING VIRUSES. In 1911,  Peyton Rous demonstrated that chicken sarcomas could be transmitted from one chicken to another using cell free filtrates of the original tumours,  thus establishing the viral etiology of this malignancy.  The RSV has been the subject of intense investigation for many years,  these studies have revealed that the oncogenic properties of this virus can be attributed to a single viral gene,  termed src Other avian retroviruses since have been isolated that induce a wide variety of sarcomas and other malignant diseases in chickens,  including erythroblastosis,  myelocytomatosis,  and myeloblastosis In addition,  many retroviruses isolated from other species,  such as cats,  mice,  and monkeys,  induce malignant sarcomas and Iymphomas.

Nonacute,  nondefective mouse retroviruses can give rise to acute,  defective transforming viruses if passed multiple times in a susceptible host (cg,  a mouse or rat) This will give rise to an occasional malignant tumour that often yields a new,  highly oncogenic (and defective) virus.  These new viruses exhibit the properties of the acute transforming viruses,  readily inducing disease in the natural host and causing transformation of cells in culture.  From a wide variety of experimental results,  it is now known that each of these acute transforming viruses has acquired a novel gene from the host cell that is responsible for the malignant properties Such genes are collectively termed oncogenes and,  for each virus,  the oncogene is designated by a three letter acronym denoting its origin.  The properties of individual oncogenes arc discussed later. Examination of this table reveals several interesting facts. For example,  the same oncogene sequence has been acquired by two different retroviruses from different species.  The fes oncogene of feline sarcoma virus is structurally identical to the fps oncogene of the avian retrovirus Fujinami sarcoma virus.  Therefore,  it can be concluded that the oncogenic potential of these genes can be manifested equally well in different species In addition,  some viruses have acquired two oncogenes (eg,  the erb A and erb B oncogenes of avian erythroblastosis virus) A growing body of evidence suggests that the two oncogenes provide a synergistic effect in the animal for the rapid and efficient outgrowth of tumour cells.

ORIGIN OF VIRAL ONCOGENES. Two major questions arose immediately on identification of the first retroviral oncogenes (1) Where do such transforming genes come from? (2) How have retroviruses acquired them? The first question was answered when investigators prepared highly radioactive DNA probes complementary to the viral oncogene sequence When these probes were hybridized to normal cell DNA,  it was observed that all normal DNA from birds,  rodents,  and humans contained one or two copies of a gene virtually identical to the oncogene under investigation Such experiments,  coupled with more sophisticated molecular cloning and DNA sequencing experiments,  have now shown that viral oncogenes have normal cell counterparts(termed protooncogenes) in the DNA of all vertebrate species.  Hence,  it is generally accepted that protooncogenes encode normal cellular proteins that perform some essential function during the lifetime of a specific cell.

If protooncogenes encode normal cellular proteins,  how are these genes acquired by the retrovirus genome,  and what alterations in the structure of the gene convert it to an oncogenic element? First,  although definitive data regarding the mechanism of oncogene “capture” do not exist,  an educated guess can be made as to how this process might take place.  Because nonacute retroviruses are ubiquitous iature,  it is postulated that,  rarely,  a nondefective virus may integrate adjacent to a protooncogene sequence.  A deletion of chromosomal DNA then could result in the joining of part of the retrovirus genome with the coding sequences of the protooncogene.   Transcription of the fused genes could readily produce a hybrid RNA containing both retrovirus sequences and protooncogene sequences.  Such an RNA molecule can undergo recombination with an existing nonacute leukemia virus transcript,  thereby yielding an RNA species similar in structure to the known acute transforming virus genome.  A comparative analysis of the DNA sequences of viral oncogenes and their cellular homologues has revealed interesting structural modifications in viral oncogene sequences.  In general,  when the proto-oncogene is captured by the retrovirus,  only a portion of the proto-oncogene is found in the virus.  In those instances in which the entire proto-oncogene is captured,  there often are multiple mutations within the captured viral gene.  Therefore,  it has been suggested that the oncogenic potential exhibited by the viral oncogene often is due to the fact that only a portion of the gene is present in the virus,  or to the fact that the vv hole gene that is present is mutated in a specific manner that alters its functional activity.

It is clear from many studies that proto-oncogene activation is a complex event (or set of events),  and considerable investigation is required before the details of such processes can be understood.  It also is clear that the biochemical events mediated by viral oncogene products can differ,  perhaps substantially,  from the biochemical events mediated by their normal cellular counterparts.

NONDEFECTIVE,  NONACUTE RNA TUMOR VIRUSES. Nonacute viruses contain the three essential genes (gag,  pol,  and env) required for replication (Fig 6) and,  when used to infect cells in culture,  these viruses replicate efficiently.  However,  they do not induce distinguishable morphologic changes in the cells Introduction of these viruses into a susceptible host (either in a laboratory setting or by transmission iature) results in a widespread viremia.  After several months,  perhaps a year,  a variety of neoplastic diseases (eg,  lymphocytic or myeloid tumours,  erythroblastic leukemias,  osteopetrosis,  nephroblastoma,  thymic sarcomas,  or lymphosarcomas) often can be observed Examination of the cells of these tumours shows that,  in most cases,  the tumours arc monoclonal in origin (i. e.,  originating from a single transformed cell).  In the case of induction of leukemia by avian leukosis virus (ALV),  the mechanism of the oncogenic event is understood.  Analyses of ALV Iymphomas have shown that each tumour contains a portion of the ALV proviral genome integrated  adjacent to the cellular proto-neogene,  c-myc.  The integration of ALV sequence s activates the expression of the c-my proto-oncogene considerably above the levels observed iormal lymphocytes Such an “insertion” activation is a critical step in the establishment of the neoplastic lymphoma,  and insertional activation of different oneogencs can occur in different tumours.

Endogenous Retroviruses. Endogenous retroviruses can be found in virtually all-vertebrate species (including man).  For the most part expression of these sequences is tightly regulated,  and viral genes are expressed only at defined times during cellular differentiation or during the lifetime of the animal However,  in the ease of chickens and mice,  the extensive experimentation with these species has led to the development of highly inbred strains Individual strains often show a unique and definable pattern of endogenous virus expression and subsequent development of malignant disease.  For example,  in some strains of mice,  such as AKR mice,  the endogenous mouse leukemia virus is activated soon after birth.  These animals develop acute viremia,  and within 6 to 12 months,  a high percentage develop leukaemia Other strains of mice (low incidence strains) may fail to acquire leukemia until late in life,  it at all High incidence strains of mice contain two dominant loci(kV 1 and kV 2) that code for the ecotropic viruses AKV 1 and AKV 2.  Expression of either locus results in expression of the leukemia virus and the induction of nepotistic disease.  In low incidence strains of mice,  resistance to leukemia results from the host’s resistance to infention by his own endogenous virus Some mouse strains do not develop leukemia,  these mice lack the endogenous leukemia virus loci.

In addition to endogenous viruses that are induced naturally and will replicate (hence the designation,  eco-tropic viruses’) in the host,  most animal cells contain endogenous retroviruses that can be induced to replicate only in cells different from the natural host.  For example,  treatment of mouse cells in culture with inhibitors of protein synthesis,  such as halogenated deoxynbonucleosides,  or inhibitors of nucleic acid synthesis induces viruses that do not grow on mouse cells but that replicate on cells of a different species.  These viruses are termed xenotropic viruses,  and their host range (ic,  the cells in which they will replicate) is determined by the ability of the env glycoprotcm to bind specifically to the appropriate cellular receptors.

Considerable interest has centered on the role of the readily inducible ecotropic mouse virus in the development of thymic lymphomas It has become increasingly clear that the ecotropic virus itself is not responsible for the induction of malignant disease However,  isolation of virus from late preleukemic or leukemic AKR mice has revealed several new viruses that have the properties of both ecotropic and xenotropic endogenous viruses.  These viruses can multiply in both mouse cells and mink cells,  and have been termed dual tropic,  or MCF (mink cell focus-forming) viruses These viruses are not endogenous viruses in the sense that their composite genomes are not present in the germline DNA of the host However,  these novel recombinant viruses appear to exhibit a unique tissue-cell tropism,  and they are important elements in establishing malignant disease The precise mechanism by which these viruses mediate cellular transformation  most certainly involves the insertional activation of cellular oncogene sequences by the integrated viruses.

Murine Mammary Tumor Viruses. In 1936,  the first murine mammary tumour viruses (MMTVs) were discovered in the milk of a strain of mice showing a high incidence of mammary carcinomas.  MMTVs are generally similar to the other RNA tumorviruses,  but their dense nucleocapsid is slightly off-center in the spherical virion; hence,  they are called B-type particles).

MMTV represents a class of mouse endogenous viruses that arc expressed in different inbred strains of mice in varying degrees In C3H mice,  MMTV is highly oncogenie and is expressed at high levels in most tissues of the mouse The virus also is found in large amounts in lactating mammary tissue and in milk Transfer of the virus to the offspring through the milk results in a high incidence of mammary carcinomas within 6 to 12 months.  It newborn mice of a high incidence strain are nursed by a foster mother of a low incidence strain,  few tumours develop Conversely,  if newborn mice of a low incidence strain are nursed by a foster mother producing MMTV,  those low incidence mice will acquire mammary tumours within the 6 to 12 month period.

MMTV is a nondefective virus and,  therefore,  has the usual complement of viral genes gag,  pol,  and env.  However,  expression of the MMTV provirus is regulated by both genetic and hormonal factors.  Glucocorticoids greatly enhance the level of expression of MMTV RNA through binding of the glucocorticoid receptor complex to a unique glucocorticoid response element in the MMTV LTR.  The mechanism of tumour induction is thought to resemble that of other nondefective,  nonacute leukemia viruses,  in that tumour cells contain integrated MMTV provirus at two distinct loci in the DNA of mouse mammary tumours.  These loci,  termed int 1 and int 2,  encode products that appear to be related to cellular growth factors

 

Human Retroviruses

HUMAN T-CELL LEUKEMIA VIRUSES. There was an extensive search for human leukemia viruses during the 1970s that was fraught with skepticism.  Virus like particles were observed frequently in human leukemia cells,  but several early isolates subsequently were shown to be laboratory contaminants of nonhuman origin.  However,  in 1981,  investigators in both the United States and Japan reported that a virus could be isolated from patients with certain T-cell malignancies.  This virus was isolated from several cultivated cell lines derived from malignant tissue as well as from fresh blood obtained from patients with adult 1 cell leukemia (ATL).

The virus,  designated human T cell leukemia virus I  (HTLV 1),  has now been clearly shown to be associated with 1 cell malignancies in humans HTLV-1 related T-cell leukemias are endemic to parts of Japan,  the Caribbean.  South America,  and Africa About 10% or the population in south-west Japan has antibodies to HTLV-1,  whereas seropositive individuals are rare in parts of the world where ATL is not endemic HTLV I also shows an association with a neurologic disease that is common in the endemic areas known as tropical spastic paraparesis (TSP) or HTLV-associated myelopathy (HAM),  this is a demyelinating disease characterized by the development of a progressive weakness or the leg and lower body muscles.  The link between HAM/TSP and HTLV-1 infection was first established by the serologic analysis of patients with TSP on the Caribbean islands of Martinique and Jamaica continued seroepidemiologic testing has shown that patients with HAM/TSP in Columbia,  Trinidad,  and Seychelles,  as well as in Japan,  also exhibit high incidences of HTLV-1 infections.  Thus,  the link between this disease and HTLV-1 has been elearlv established.  Although some HTLV-1 infections eventually lead to overt clinical disease in the form of ATL  or HAM/TSP,  most remain subclnical throughout the life of the infected individual.  The lifetime risk of developing disease has been estimated at only a few percent or less.

A second related virus,  designated HTLV II,  originally was isolated from a patient with T cell variant hairy-cell leukemia HTLV II has been shown to be endemic in Native American populations,  but has not been clearly linked to any human disease HTLV-I and HTLV-II can be transmitted in three different ways through sexual contact,  through contaminated blood,  and from an infected mother to her child during the perinatal period.  The last mode of transmission occurs mainly through infected cells in breast milk.  Therefore,  it is recommended that infected mothers do not breastfeed their children

Because HTLV can be spread by blood,  and thus by transfusions,  blood banks routinely test their blood supply for the presence of HTLV Intravenous drug abusers in the United States are showing an increasing prevalence of HTLV infection (mainly HTLV-II).  Many of these individuals also are infected with HIV It is unclear whether such patients progress more rapidly to the acquired immunodeficiency syndrome (AIDS),  and whether they have a higher incidence of ATL or HAM/TSP.

Structure and Replication of Human T-Cell Leukemia Viruses I and II. The genome of HTLV closely resembles that of other vertebrate retroviruses in that it encodes the three essential gone products (gag,  pol,  and env) required for virus replication (Fig.).

 

FIGURE. Genome structure of the human retroviruses human T-cell leukemia/lymphoma virus (HTLV-I,  HTLV-II),  and human immunodeficiency virus (HIV).

 

 However,  the HTLV genome contains additional genes that contribute to the ability of HTLV to autoregulate its replication in infected cells.  This distinguishes the replication cycle of this virus from that of more conventional retroviruses.  The best characterized of these genes,  designated tax and rex,  reside downstream of the env gene.  Together,  the tax and rex gene products regulate the production of HTLV RNA.  The tax protein is a strong “transactivator” of cellular and viral gene expression.  The tax protein binds to host-cell transcription factors that,  in turn,  actively promote efficient transcription of virus RNA.  The tax protein also promotes or enhances (transactivates) the expression of certain cellular genes (e. g.,  the interleukin-2 receptor).  The rex protein acts specifically to promote the expression of the viral structural proteins gag,  pol,  and env. 

 

 

The rex protein binds to an element present in the 5′ end of them RNAs for these proteins,  known as the rex response element,  and promotes the transport of the RNAs from the nucleus to the cytoplasm of infected cells.  The function of the rex protein appears to be similar to that of the HIV rev protein discussed later.  The tax and rex genes both are situated at the 3′ end of the viral genome.  This region,  previously known as the pX region,  also encodes other gene products.  The exact functions of these in viral replication are unknown.

The relationship between HTLV replication and the appearance of ATL is unclear.  Analysis of cell lines derived from patients with ATL.  has shown that each individual ATL cell line has HTLV sequences integrated at a different site in the chromosomal DNA.  No evidence for “insertional” activation of a cellular oncogene has been obtained.  What,  then,  is the mechanism for HTLV-induced disease? One model suggests that the HTLV-encoded tax gene product may act to transactivate cellular genes that,  in turn,  stimulate T-cell proliferation.  The rapid proliferation of a population of T cells then could give rise to the activation of cellular oncogenes through additional mutational mechanisms (see Chap.  44).  Evidence for this model comes from the observation that tax expression in T lymphocytes increases the level of expression of interleukin-2 and interleukin-2 receptors,  cellular components that are required for T-cell proliferation.

Much remains to be done to elucidate the role of HTLV in human malignancies and other diseases.  How-ever,  insights into the role of virus replication and the contributions of novel viral gene products will help to paint a clearer picture of virus-induced cellular changes,  both malignant and otherwise.

 

HUMAN IMMUNODEFICIENCY VIRUSES

In 1981,  a novel,  epidemic form of immunodeficiency,  termed AIDS,  was recognized.  Between 1981 and 1991,  there was a virtual explosion in the number of AIDS cases in the United States,  and this disease is now one of the leading causes of death in young individuals.  In 1981,  there were 310 cases of AIDS reported in this country and 135 deaths attributed to the disease.  In 1991 alone,  more than 40, 000 new AIDS cases were reported and more than 30, 000 people died of the disease.  AIDS is now known to be caused by a human retrovirus,  designated HIV.  The disease is characterized by opportunistic infections and malignant diseases in patients without a recognized cause for immunodeficiency.  Numerous opportunistic infections have been observed,  predominantly caused by Pneumocystis carinii,  cytomegalovirus,  atypical mycobacteria,  Toxoplasma gondii,  Candida,  herpes simplex virus,  Cryptococcus neoformans,  and Cryptosporidium.  Active tuberculosis also is seen at an increasing frequency.  Other highly distinctive features of AIDS are the occurrence of Kaposi’s sarcoma (particularly in gay men) and dementing neurologic disorders.  As many as 5% to 10% of infected individuals develop lymphomas that frequently are positive for Epstein-Barr virus.  In addition,  the incidence of cervical carcinoma is significantly increased in HIV-infected women.  AIDS is a disease of the immune system,  and a hallmark of the disease is an abnormally low number of CD4-positive cells.

Like HTLV,  HIV is transmitted by sexual contact through infected blood,  and from mother to child.  HIV can be transmitted during both pregnancy and the neonatal period,  and recent studies suggest that vertical transmission can be reduced significantly by azidothymidine(AZT) treatment of the mother during pregnancy.  With-out treatment,  about 25% of children born to infected mothers acquire the virus.  Many of these go on to rapidly develop AIDS.  The blood supply is now routinely tested for HIV,  but before tests were developed,  many individuals became infected as a result of blood transfusions.  Blood-derived products used in the treatment of haemophilia also were contaminated frequently with HIV,  and a large percentage of patients with severe hemophilia were infected early on in the epidemic.  Many of these have now died of AIDS.

In the United States and Europe,  HIV infection and,  subsequently,  AIDS still occurs mostly in certain high-risk groups.  These include gay and bisexual men,  intravenous drug abusers,  heterosexual partners of members of these groups,  and infants born to HIV-positive mothers.  AIDS is still mainly a male disease in these countries.  However,  the number (if cases in women is increasing rapidly.  In the United States,  most infected women belong to minority groups (74%),  and many women infected in recent years have reported heterosexual activity as their only risk factor.  This indicates that heterosexual transmission is be-coming more common in this country.  In many other areas of the world,  the disease already is spread primarily by heterosexual transmission and affects men and women in equal proportions.  In Africa,  where the epidemic is thought to have originated,  as many as 10 million people were infected with HIV by mid-1994.  It is unclear how many already have died of AIDS,  because most cases are not reported,  but the estimated figure is 2. 5 million (50% women).  Latin America also has a serious problem,  with millions of infected individuals.  HIV infection also is spreading rapidly in parts of Asia,  especially in Thailand and India,  and it is clear that AIDS soon will be a serious problem in these countries.  It has been estimated that about 1 million individuals in the United States are infected with HIV.  Many of these have no symptoms,  and many do not know that they are infected.  By mid-1994, the total number of reported AIDS cases in the United States alone had reached almost 400, 000,  and about 60% of these patients already had died from the disease.

The initial isolation of HIV from the cells of patients with AIDS was reported by a group of French scientists in 1983.  This was followed by the isolation and continuous propagation of other isolates by scientists in both France and the United States in 1984,  clearly documenting the link between the virus and AIDS.  Several lines of evidence have now established HIV as the main etiologic agent for AIDS.  Infected individuals often remain free of symptoms for many years.  Recent estimates indicate that 80% to 90% of those infected go on to develop AIDS within 10 to 12 years of infection.  The time for progression to AIDS varies greatly.  Long-term survivors (i.e.,  infected patients who have been observed for 7 years or more) include individuals with normal levels of CD4-positive cells.  Some of these may never go on to develop AIDS. In other patients,  levels of CD4-positive cells drop dramatically within years after infection,  leading to rapid development of the disease and death.  The factors that determine these different outcomes are still largely unknown.

Human Immunodeficiency Virus Genome Structure.

Human immunodeficiency virus belongs to the Lentivirus subfamily.  Lentiviruses are characterized by a complex genome structure with several more genes in addition to gag,  pol,  and env.  They also are characterized by their efficient replication and their ability to cause a lytic infection (i.e.,  an infection that kills the host cell).  It is now recognized that there are two different types of HIV (HIV-1 and HIV-2).  HIV-2 is most prevalent in parts of West Africa,  and only a few cases of HIV-2 infection have been reported in the United States.

Human Immunodeficiency Virus Genome Structure.

Human immunodeficiency virus belongs to the Lentivirus subfamily.  Lentiviruses are characterized by a complex genome structure with several more genes in addition to gag,  pol,  and env.  They also are characterized by their efficient replication and their ability to cause a lytic infection (i.e.,  an infection that

 n.wikipedia.org/wiki/HIV

HIV is a member of the genus Lentivirus,^[6] part of the family of Retroviridae.^[7] Lentiviruses have many morphologies and biological properties in common. Many species are infected by lentiviruses, which are characteristically responsible for long-duration illnesses with a long incubation period.^[8] Lentiviruses are transmitted as single-stranded, positive-sense, enveloped RNA viruses. Upon entry into the target cell, the viral RNA genome is converted (reverse transcribed) into double-stranded DNA by a virally encoded reverse transcriptase that is transported along with the viral genome in the virus particle. The resulting viral DNA is then imported into the cell nucleus and integrated into the cellular DNA by a virally encoded integrase and host co-factors.^[9] Once integrated, the virus may become latent, allowing the virus and its host cell to avoid detection by the immune system. Alternatively, the virus may be transcribed, producing new RNA genomes and viral proteins that are packaged and released from the cell as new virus particles that begin the replication cycle anew.

Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is the virus that was initially discovered and termed both LAV and HTLV-III. It is more virulent, more infective,^[10] and is the cause of the majority of HIV infections globally. The lower infectivity of HIV-2 compared to HIV-1 implies that fewer of those exposed to HIV-2 will be infected per exposure. Because of its relatively poor capacity for transmission, HIV-2 is largely confined to West Africa.^[11]

Classification

See also: Subtypes of HIV

Comparison of HIV species
Species	Virulence	Infectivity	Prevalence	Inferred origin
HIV-1	High	High	Global	Common Chimpanzee
HIV-2	Lower	Low	West Africa	Sooty Mangabey

Structure and genome

Main article: Structure and genome of HIV

The HIV genome has been shown to contain at least six extra genes.  Three of these genes (tat,  rev,  and nef) encode regulatory proteins that are likely to play important roles in viral pathogenesis.  The HIV-1 genome contains three additional accessory genes (vpu,  vif,  and vpr) that are dispensable for replication in some tissue-culture cells.  The HIV-2 genome differs from HIV-1 in that the vpu gene is missing. 

However,  the HIV-2 genome contains a gene (vpx) that is not present in HIV-1.  The exact role of the accessory gene products in virus replication is unclear.

The tat gene plays a major role in the regulation viral gene expression,  and its expression is essential virus growth.  The tat protein is an 82-amino acid protein found in the nucleus of infected cells.  The tat gene contains two coding exons interrupted by an intron,  and the virus RNA has to be multiply spliced to generate the mRNA for this protein.  The tat gene product (like the HTLV-tax) gene product) is a powerful transactivator viral transcription.  Tat functions to enhance virus RNA transcription by specifically interacting with sequence the 5′ end of the viral genome,  the TAR (tat response) sequences.  The TAR sequences are the first sequences to be transcribed from the viral promoter.  The newly transcribed TAR RNA forms a stem-loop structure that specifically binds the tat protein.  This promotes elongation of the RNA chain and probably also initiatioew RNA synthesis.  Thus,  TAR acts as an enhancer the RNA level.  In the presence of tat,  the amounts full-length viral transcripts are increased several hundred-fold.  Hence,  in an infected cell,  the presence or absence of the tat protein has marked effects on the efficiency of virus transcription.

The rev protein also is made from a multiply spliced mRNA.  This protein functions similarly to the HTLrex protein.  Rev (ATL 6-amino acid protein in HIV promotes the transport from the host-cell nucleus to the cytoplasm of the mRNAs encoding the structural proteins gag,  gag/pol,  and env,  as well as the mRNAs for vif,  vpr and vpu.  In the absence of rev,  only the nef,  rev,  and mRNAs reach the cytoplasm.  The rev-regulated mRNAs all are incompletely spliced and contain complete intro

The nef protein is dispensable for virus replication most tissue-culture cells.  However,  nef is likely to pan important role in pathogenesis.  The nef protein down-regulates the CD4 receptor and also may affect cellular signal transduction pathways.

Human Immunodeficiency Virus Replication and Pathogenesis. The basis for the immunopathogenesis of HIV infection is a severe depletion of the helper/inducer subset of T lymphocytes expressing the CD4 marker.  This depletion causes a severe combined immunodeficiency,  because the T4 lymphocytes play a central role in the immune response to foreign antigens.

 

 

 

                                 Figure. Structure of HIV.

 

Human Immunodeficiency Virus Replication and Pathogenesis. The basis for the immunopathogenesis of HIV infection is a severe depletion of the helper/inducer subset of T lymphocytes expressing the CD4 marker.  This depletion causes a severe combined immunodeficiency,  because the T4 lymphocytes play a central role in the immune response to foreign antigens.

 

 

HIV’s tropism for T4 lymphocytes reflects the utilization of the CD4 molecule as a high-affinity receptor for the virus (fig.). 

 

FIGURE. The entry of the human immunodeficiency virus into T helper celts involves Hiv gpl20 binding to CD4 receptor of the T cell with CD26 -isbibtance for entry.

 

 

HIV is different in structure from other retroviruses. It is roughly spherical^[12] with a diameter of about 120 nm, around 60 times smaller than a red blood cell, yet large for a virus.^[13] It is composed of two copies of positive single-stranded RNA that codes for the virus’s nine genes enclosed by a conical capsid composed of 2,000 copies of the viral protein p24.^[14] The single-stranded RNA is tightly bound to nucleocapsid proteins, p7, and enzymes needed for the development of the virion such as reverse transcriptase, proteases, ribonuclease and integrase. A matrix composed of the viral protein p17 surrounds the capsid ensuring the integrity of the virion particle.^[14]

This is, in turn, surrounded by the viral envelope that is composed of two layers of fatty molecules called phospholipids taken from the membrane of a human cell when a newly formed virus particle buds from the cell. Embedded in the viral envelope are proteins from the host cell and about 70 copies of a complex HIV protein that protrudes through the surface of the virus particle.^[14] This protein, known as Env, consists of a cap made of three molecules called glycoprotein (gp) 120, and a stem consisting of three gp41 molecules that anchor the structure into the viral envelope.^[15] This glycoprotein complex enables the virus to attach to and fuse with target cells to initiate the infectious cycle.^[15] Both these surface proteins, especially gp120, have been considered as targets of future treatments or vaccines against HIV.^[16]

The RNA genome consists of at least seven structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, and INS), and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion of tat env and rev), encoding 19 proteins. Three of these genes, gag, pol, and env, contain informatioeeded to make the structural proteins for new virus particles.^[14] For example, env codes for a protein called gp160 that is broken down by a cellular protease to form gp120 and gp41. The six remaining genes, tat, rev, nef, vif, vpr, and vpu (or vpx in the case of HIV-2), are regulatory genes for proteins that control the ability of HIV to infect cells, produce new copies of virus (replicate), or cause disease.^[14]

The two Tat proteins (p16 and p14) are transcriptional transactivators for the LTR promoter acting by binding the TAR RNA element. The TAR may also be processed into microRNAs that regulate the apoptosis genes ERCC1 and IER3.^[17][18] The Rev protein (p19) is involved in shuttling RNAs from the nucleus and the cytoplasm by binding to the RRE RNA element. The Vif protein (p23) prevents the action of APOBEC3G (a cell protein that deaminates DNA:RNA hybrids and/or interferes with the Pol protein). The Vpr protein (p14) arrests cell division at G2/M. The Nef protein (p27) down-regulates CD4 (the major viral receptor), as well as the MHC class I and class II molecules.^[19][20][21]

Nef also interacts with SH3 domains. The Vpu protein (p16) influences the release of new virus particles from infected cells.^[14] The ends of each strand of HIV RNA contain an RNA sequence called the long terminal repeat (LTR). Regions in the LTR act as switches to control production of new viruses and can be triggered by proteins from either HIV or the host cell. The Psi element is involved in viral genome packaging and recognized by Gag and Rev proteins. The SLIP element (TTTTTT) is involved in the frameshift in the Gag-Pol reading frame required to make functional Pol.^[14]

Tropism

The term viral tropism refers to which cell types HIV infects. HIV can infect a variety of immune cells such as CD4⁺ T cells, macrophages, and microglial cells. HIV-1 entry to macrophages and CD4⁺ T cells is mediated through interaction of the virion envelope glycoproteins (gp120) with the CD4 molecule on the target cells and also with chemokine coreceptors.^[15]

Macrophage (M-tropic) strains of HIV-1, or non-syncitia-inducing strains (NSI) use the β-chemokine receptor CCR5 for entry and are, thus, able to replicate in macrophages and CD4⁺ T cells.^[22] This CCR5 coreceptor is used by almost all primary HIV-1 isolates regardless of viral genetic subtype. Indeed, macrophages play a key role in several critical aspects of HIV infection. They appear to be the first cells infected by HIV and perhaps the source of HIV production when CD4⁺ cells become depleted in the patient. Macrophages and microglial cells are the cells infected by HIV in the central nervous system. In tonsils and adenoids of HIV-infected patients, macrophages fuse into multinucleated giant cells that produce huge amounts of virus.

T-tropic isolates, or syncitia-inducing (SI) strains replicate in primary CD4⁺ T cells as well as in macrophages and use the α-chemokine receptor, CXCR4, for entry.^[22][23][24] Dual-tropic HIV-1 strains are thought to be transitional strains of HIV-1 and thus are able to use both CCR5 and CXCR4 as co-receptors for viral entry.

The α-chemokine SDF-1, a ligand for CXCR4, suppresses replication of T-tropic HIV-1 isolates. It does this by down-regulating the expression of CXCR4 on the surface of these cells. HIV that use only the CCR5 receptor are termed R5; those that use only CXCR4 are termed X4, and those that use both, X4R5. However, the use of coreceptor alone does not explain viral tropism, as not all R5 viruses are able to use CCR5 on macrophages for a productive infection^[22] and HIV can also infect a subtype of myeloid dendritic cells,^[25] which probably constitute a reservoir that maintains infection when CD4⁺ T cell numbers have declined to extremely low levels.

Some people are resistant to certain strains of HIV.^[26] For example, people with the CCR5-Δ32 mutation are resistant to infection with R5 virus, as the mutation stops HIV from binding to this coreceptor, reducing its ability to infect target cells.

Sexual intercourse is the major mode of HIV transmission. Both X4 and R5 HIV are present in the seminal fluid, which is passed from a male to his sexual partner. The virions can then infect numerous cellular targets and disseminate into the whole organism. However, a selection process leads to a predominant transmission of the R5 virus through this pathway.^[27][28][29] How this selective process works is still under investigation, but one model is that spermatozoa may selectively carry R5 HIV as they possess both CCR3 and CCR5 but not CXCR4 on their surface^[30] and that genital epithelial cells preferentially sequester X4 virus.^[31] In patients infected with subtype B HIV-1, there is often a co-receptor switch in late-stage disease and T-tropic variants appear that can infect a variety of T cells through CXCR4.^[32] These variants then replicate more aggressively with heightened virulence that causes rapid T cell depletion, immune system collapse, and opportunistic infections that mark the advent of AIDS.^[33] Thus, during the course of infection, viral adaptation to the use of CXCR4 instead of CCR5 may be a key step in the progression to AIDS. A number of studies with subtype B-infected individuals have determined that between 40 and 50 percent of AIDS patients can harbour viruses of the SI and, it is presumed, the X4 phenotypes.^[34][35]

HIV-2 is much less pathogenic than HIV-1 and is restricted in its worldwide distribution. The adoption of “accessory genes” by HIV-2 and its more promiscuous pattern of coreceptor usage (including CD4-independence) may assist the virus in its adaptation to avoid innate restriction factors present in host cells. Adaptation to use normal cellular machinery to enable transmission and productive infection has also aided the establishment of HIV-2 replication in humans. A survival strategy for any infectious agent is not to kill its host but ultimately become a commensal organism. Having achieved a low pathogenicity, over time, variants more successful at transmission will be selected.^[36]

 

Replication cycle

 

HIV enters macrophages and CD4⁺ T cells by the adsorption of glycoproteins on its surface to receptors on the target cell followed by fusion of the viral envelope with the cell membrane and the release of the HIV capsid into the cell.^[37][38]

Entry to the cell begins through interaction of the trimeric envelope complex (gp160 spike) and both CD4 and a chemokine receptor (generally either CCR5 or CXCR4, but others are known to interact) on the cell surface.^[37][38] gp120 binds to integrin α₄β₇ activating LFA-1 the central integrin involved in the establishment of virological synapses, which facilitate efficient cell-to-cell spreading of HIV-1.^[39] The gp160 spike contains binding domains for both CD4 and chemokine receptors.^[37][38]

The first step in fusion involves the high-affinity attachment of the CD4 binding domains of gp120 to CD4. Once gp120 is bound with the CD4 protein, the envelope complex undergoes a structural change, exposing the chemokine binding domains of gp120 and allowing them to interact with the target chemokine receptor.^[37][38] This allows for a more stable two-pronged attachment, which allows the N-terminal fusion peptide gp41 to penetrate the cell membrane.^[37][38] Repeat sequences in gp41, HR1, and HR2 then interact, causing the collapse of the extracellular portion of gp41 into a hairpin. This loop structure brings the virus and cell membranes close together, allowing fusion of the membranes and subsequent entry of the viral capsid.^[37][38]

After HIV has bound to the target cell, the HIV RNA and various enzymes, including reverse transcriptase, integrase, ribonuclease, and protease, are injected into the cell.^[37] During the microtubule-based transport to the nucleus, the viral single-strand RNA genome is transcribed into double-strand DNA, which is then integrated into a host chromosome.

HIV can infect dendritic cells (DCs) by this CD4-CCR5 route, but another route using mannose-specific C-type lectin receptors such as DC-SIGN can also be used.^[40] DCs are one of the first cells encountered by the virus during sexual transmission. They are currently thought to play an important role by transmitting HIV to T-cells when the virus is captured in the mucosa by DCs.^[40] The presence of FEZ-1, which occurs naturally in neurons, is believed to prevent the infection of cells by HIV.^[41]

Replication and transcription

Shortly after the viral capsid enters the cell, an enzyme called reverse transcriptase liberates the single-stranded (+)RNA genome from the attached viral proteins and copies it into a complementary DNA (cDNA) molecule.^[42] The process of reverse transcription is extremely error-prone, and the resulting mutations may cause drug resistance or allow the virus to evade the body’s immune system. The reverse transcriptase also has ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that creates a sense DNA from the antisense cDNA.^[43] Together, the cDNA and its complement form a double-stranded viral DNA that is then transported into the cell nucleus. The integration of the viral DNA into the host cell’s genome Other cells that express the CD4 molecule, such as macrophages and monocytes,  also are targets for HIV infection.  After binding of the HIV SU protein  gpl20 to the CD4 molecule,  fusion of viral and cellumembranes enables the virus to enter the host cell.  The  fusion is mediated by the transmembrane portion of the env protein (gp41).  After the core is internalized,  the HIV genome is transcribed to DNA,  and proviral DNA is integrated into the host chromosome.  After integration of the provirus,  the infection can become latent or virus replication can be initiated.  The frequency at which a latent infection rather than a productive infection is established is unclear.  However,  it is known that a large number of cells are actively replicating virus even during the clinical latency period.  The precise relationship between cell activation and virus replication has not been delineated, but the extent of antigenic stimulation of the immune system may play a role in determining the period betwee nvirus infection and severe T4-cell depletion.

Several mechanisms have been proposed to explain the depletion of the CD4 cells.  In addition to cells being killed directly by HIV replication,  cells expressing viral envelope protein on the cell surface may interact with uninfected CD4-bearing cells,  thereby promoting the fusion of infected and uninfected T4 lymphocytes.  This might lead to the death of noninfected cells.  In addition, cell-cell fusion could promote efficient spread of virus from cell to cell.  Immune mechanisms also may play an important role in T4-cell depletion.  For example,  infected T4 cells expressing envelope protein on their surface maybe recognized as nonself and efficiently cleared from the system.  In addition,  binding of circulating gpl20 to the surface of uninfected T4 cells also may designate these cells as nonself,  leading to their destruction.

The importance of macrophages and monocytes in the course of HIV infection and disease is becoming more evident.  Monocytes can be infected with HIV in vitro, and virus can be isolated from monocytes obtained from the blood and organs of HIV-infected individuals,  indicating that monocytes serve as a major reservoir for HIV in the body.  The infected monocyte may transport the virus to various organs of the body.  Macrophages also may contribute to the unique neurologic symptoms associated with AIDS,  because they are the major cell type harbouring HIV in the brain,  giving rise to the speculation that these cells contribute to the spread of virus within the central nervous system.

It has been postulated that antigenic stimulation of an infected individual’s immune system might stimulate T-cell proliferation,  thereby promoting virus spread.  In addition,  it is possible that concomitant virus infections(i.e.,  Epstein-Barr virus,  cytomegalovirus,  hepatitis B,  or herpes simplex virus) can induce HIV expression,  leading to more rapid disease progression.

 

Assembly and release

The final step of the viral cycle, assembly of new HIV-1 virions, begins at the plasma membrane of the host cell. The Env polyprotein (gp160) goes through the endoplasmic reticulum and is transported to the Golgi complex where it is cleaved by furin resulting in the two HIV envelope glycoproteins, gp41 and gp120.^[46] These are transported to the plasma membrane of the host cell where gp41 anchors gp120 to the membrane of the infected cell. The Gag (p55) and Gag-Pol (p160) polyproteins also associate with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud from the host cell. The budded virion is still immature as the gag polyproteins still need to be cleaved into the actual matrix, capsid and nucleocapsid proteins. This cleavage is mediated by the also packaged viral protease and can be inhibited by antiretroviral drugs of the protease inhibitor class. The various structural components then assemble to produce a mature HIV virion.^[47] Only mature virions are then able to infect another cell.

The pathogenesis of AIDS is dependent on the biology of HIV, e.g:

 http://www.google.com.ua/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&sqi=2&ved=0CEIQFjAB&url=http%3A%2F%2Fwww.mcb.uct.ac.za%2Fcann%2F335%2FAIDSI.html&ei=mZlAUfWpGM3Aswam2IDYDQ&usg=AFQjCNF0nq_PMjasEX-fs4QlduDq1Mhw8g&sig2=rj8Uzh3NsYJNgMIxOkQDAA&bvm=bv.43287494,d.Yms&cad=rja

• ‘Trojan horse’ mechanism – virus escapes recognition by replication inside monocytes, from where it can spread to other tissues and other hosts.

• Latency – Lentiviruses do not show true latency (unlike Herpes viruses or lambda) but do have the capacity to control the expression of their genome by means of virus-encoded trans-acting regulatory proteins (tat and rev).

• Antigenic variation – new variants continually arise. In other Lentiviruses such as CAEV, each new antigenic variant results in a flare up of disease. May also occur in HIV and contribute to decline of immune system – ‘ratcheting’ mechanism due to increasing virus load?

Some of the immune abnormalities in HIV infection include:

• Altered cytokine expression

• Decreased CTL and NK cell function

• Decreased humoral and proliferative response to antigens and mitogens

• Decreased MHC-II expression

• Decreased monocyte chemotaxis

• Depletion of CD4+ cells

• Impaired DTH reactions

• Lymphopenia

• Polyclonal B-cell activation

It is not clear how much of the pathology of AIDS is directly due to the virus and how much is caused by the immune system itself. There are numerous models which have been suggested to explain how HIV causes immune deficiency:

Direct Cell Killing:

This was the first mechanism suggested, based on the behavior of certain laboratory isolates of HIV. Subsequent experiments suggested that there is not sufficient virus present in AIDS patients to account for all the damage seen, although killing of CD4+ cells may contribute to the overall pattern of pathogenesis seen in AIDS. Indirect effects of infection, e.g. disturbances in cell biochemistry and lymphokine production may also affect the regulation of the immune system:

However, the expression of virus antigens on the surface of infected cells leads to indirect killing by the immune system (NK/CTL/ADCC) – effectively a type of autoimmunity. Recently, this hypothesis has been resurrected as a result of more accurate quantitation of virus load and replication kinetics in infected individuals (see below).

Antigenic Diversity:

This theory holds that the continual generation of new antigenic variants eventually swamps and overcomes the immune system, leading to its collapse.
There is no doubt that new antigenic variants of HIV constantly arise during the long course of AIDS, in a similar way to CAEV infection of goats. It has not been completely established how this might lead to the collapse of the immune system, but it is envisaged that there might be a ‘ratchet’ effect, with each new variant contributing to the slight but irreversible decline in immune function.
N.B: Because of the way virus infections are handled by the immune system, it is probable that variation of T-cell epitopes on target proteins recognised by CTLs are at least (probably more) important than B-cell epitopes which generate the antibody response to a foreign antigen. It has recently been reported that at least some variants can inhibit the CTL response to wild-type HIV (Meier, U. et al. Cytotoxic T lymphocyte lysis inhibited by viable HIV mutants. Science 270: 1360-62, 1995).
A mathematical model has been constructed which simulates antigenic variation during the course of infection. When primed with all of the known data about the state of immune system during HIV infection, it provides a startling accurate depiction of the course of AIDS (Nowak MA, et al. Antigenic diversity thresholds and the development of AIDS. Science 254: 963-969, 1991).
Medscape Article: Genotypic Variation and Molecular Epidemiology of HIV.

The Superantigen Theory:

Superantigens are molecules which short-circuit the immune system, resulting in massive activation of T-cells rather than the usual, carefully controlled response to foreign antigens. It is believed that they do this by binding to both the variable region of the beta-chain of the T-cell receptor (V-beta) and to MHC II molecules, cross-linking them in a non-specific way:

This results in polyclonal T-cell activation rather than the usual situation where only the few clones of T-cells responsive to a particular antigen presented by the MHC II molecule are activated. The over-response of the immune system produced results in autoimmunity, as rare clones of T-cells which recognise self antigens are activated, and immune suppression, as the activated cells subsequently die or are killed by other activated T-cells. It is possible that such superantigens might also induce apoptosis (pronounced “apo-tosis”), or ‘programmed cell killing’ (Cohen JJ. Apoptosis. Immunol. Today 14: 126-136, 1993):

It has been reported that in some AIDS patients, certain clones of T-cells bearing particular Vb T-cell receptor rearrangements are depleted or absent. This is precisely what would be expected if some clones of cells were being eliminated by the presence of a superantigen. However, unlike other retroviruses (e.g. mouse mammary tumour virus (MMTV) and the murine leukaemia virus (MuLV) responsible for murine acquired immunodeficiency syndrome [MAIDS]) no superantigen has been conclusively identified in HIV, despite intensive investigation. Thus the practical relevance of superantigens in AIDS remains in some doubt. However, it is possible that exposure to superantigens produced by opportunistic infection(s) might play an important role in AIDS.

Receptor Signalling:

There have been several reports that HIV binding to CD4 induces an intracellular signal which may have a detrimental effect on cells. None of these have been completely convincing. However, there is a second component to the HIV receptor which is required for cell entry: chemokine receptors such as CXCR4 & CCR5. Although HIV-mediated signal transduction is not required for fusion & entry, at least some HIV isolates induce a signal when the envelope protein binds to CCR5 (Weissman et al, Nature 389: 981-985, 1997). HIV disease is characterized (in part) by persistent immune activation. Envelope-mediated signalling through binding to chemokine receptors could contribute to cellular activation. HIV replicates only in activated cells, so this activity promotes replication directly & may also assist in the spread of the virus to uninfected cells by inducing the migration of activated cells to sites of virus replication via chemotaxis. These signals may also contribute indirectly to the pathogenesis of the infection by inducing apoptosis or anergy.
Neuronal apoptosis is a feature of HIV-1 infection in the brain, contributing to dementia. gp120 from some strains of HIV binds with high affinity to CXCR4 expressed on hNT neurons. Both gp120 and the Cys-X-Cys chemokine SDF-1[alpha] can directly induce apoptosis in hNT neurons in the absence of CD4 and in a dose-dependent manner. Thus the HIV-1 envelope glycoprotein may elicit apoptotic responses through chemokine receptors.

TH1-TH2 Switch:

Early in HIV infection, TH1-responsive T-cells predominate and are effective in controlling (but not eliminating) the virus. At some point, a (relative) loss of the TH1 response occurs and TH2 HIV-responsive cells predominate:

The hypothesis is therefore that the TH2-dominated humoral response is not effective at maintaining HIV replication at a low level and the virus load builds up, resulting in AIDS.

N.B. This is a theoretical proposal, and has not yet been proved, but is shaping our understanding of the immune response to many different pathogens, not just HIV (Clerici M, Shearer G. A TH1-TH2 switch is a critical step in the etiology of HIV infection. Immunol. Today 14: 107-111, 1993).

Virus Load and Replication Kinetics:

Recent reports involving accurate quantitation of the amount of virus in infected patients have revealed that much more virus is present than originally thought. Using quantitative PCR methods to accurately measure the amount of virus present in HIV-infected individuals and determining how these levels change when patients are treated with compounds which inhibit virus replication, it has been shown that:

• Continuous and highly productive replication of HIV occurs in all infected individuals, although the rates of virus production vary by up to 70-fold in different individuals

• The average half-life of an HIV particle/infected cell in vivo is 2.1 days

• Up to 2×10⁹ HIV particles are produced each day

• An average of 2.6×10⁹ new CD4+ cells are produced

Thus, contrary to what has recently been thought, there is a very dynamic situation in HIV-infected subjects involving continuous infection, destruction and replacement of CD4+ cells, with billions of new cells being infected and killed each day. These data suggest a return to cellular killing (although predominantly immune-mediated rather than virus-mediated) as a direct cause of the CD4+ cell decline in AIDS. For reasons which are not yet clear, this is a (marathon) race between virus production, destruction and cellular regeneration which, after many years, most individuals loose, resulting in the absolute decline of the CD4 segment of the immune system and the development of full-blown AIDS.

Conclusions:

The ultimate mechanism by which HIV infection causes AIDS remains unknown, but recent reports strongly implicate immune-mediated killing of virus-infected cells as the major factor in the pathogenesis of this disease. These new ideas are informing future thinking about possible therapeutic intervention in HIV-infected individuals.

Therapy of HIV Infection:

Several distinct classes of drugs are now used to treat HIV infection:

1. Nucleoside-Analog Reverse Transcriptase Inhibitors (NRTI). These drugs inhibit viral RNA-dependent DNA polymerase (reverse transcriptase) and are incorporated into viral DNA (they are chain-terminating drugs).

• Zidovudine (AZT = ZDV, Retrovir) first approved in 1987

• Didanosine (ddI, Videx)

• Zalcitabine (ddC, Hivid)

• Stavudine (d4T, Zerit)

• Lamivudine (3TC, Epivir)

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). In contrast to NRTIs, NNRTIs are not incorporated into viral DNA; they inhibit HIV replication directly by binding non-competitively to reverse transcriptase.

• Nevirapine (Viramune)

• Delavirdine (Rescriptor)

Protease Inhibitors. These drugs are specific for the HIV-1 protease and competitively inhibit the enzyme, preventing the maturation of virions capable of infecting other cells.

• Saquinavir (Invirase) first approved in 1995

• Ritonavir (Norvir)

• Indinavir (Crixivan)

• Nelfinavir (Viracept)

 

Immune activation and AIDS pathogenesis

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The mechanisms by which HIV induces the immune dysfunction clinically defined as AIDS have been a subject of intense study since the discovery of the virus in the early 1980s. Initial virological analysis demonstrated low levels of virus replication in infected patients, suggesting that the virus alone was incapable of inducing AIDS and that additional factors must also play a role in determining the HIV-associated immunologic dysfunction. This concept has recently been emphasized from a statistical perspective by the observation that while the level of HIV replication is significantly correlated with the risk of disease progression ^[1], this parameter only predicts a minor part of the variation in the rate of progression among infected patients ^[2]. In the mid 1990s, improvements in the techniques available to detect HIV demonstrated that virus replication was active throughout the course of the disease ^[3,4]. In addition, the observation that inhibition of viral replication with antiretroviral drugs substantially attenuates disease progression established very clearly that virus replication is responsible for pathogenicity. What remains poorly defined, however, is the mechanistic linkage between virus replication and the onset of AIDS.

A model of CD4 T-cell depletion based entirely on direct virus infection and killing of these cells was put forward in the mid 1990s ^[5,6]. This so-called ‘tap-and-drain’ model proposed that progression to AIDS in HIV-infected individuals resulted from a failure of the immune system’s homeostatic response to keep up with a high rate of loss of CD4 T cells ^[5,6]. The model offered an explanation for the rapid increase of CD4 T-cell counts following inhibition of virus replication by antiretroviral therapy. However, this model and its later versions ^[7,8] were challenged on theoretical and experimental grounds as they did not appear to grasp the complexity of T-cell dynamics in response to ongoing viral replication and painted a simplistic picture of AIDS pathogenesis ^[9-17].

The idea that chronic immune activation plays a major role in AIDS pathogenesis was first put forward by Ascher and Sheppard ^[18] and, in parallel – but from a rather different perspective – by Grossman and colleagues in the late 1980s/early 1990s ^[19,20]. Shortly thereafter, Giorgi and colleagues published a series of clinical studies supporting the concept that an excessive/aberrant immune activation is a fundamental driving force for the HIV-associated immune dysfunction. These studies identified the level of CD8 T-cell activation, as determined by CD38 and HLA-DR expression, as a better correlate of disease progression than viral load ^[21-24]. While exact characterization of the HIV-associated chronic immune activation remains incomplete, an activation/dysfunction phenotype is apparent for many different immune cell types in HIV infection. With regard to T-cells, the assessment of immune activation can be made through: (i) high frequency of T cells expressing markers of activation and proliferation ^[25-27]; (ii) high levels of activation-induced apoptosis of uninfected T cells ^[28-32]; (iii) high levels of T-cell proliferation as measured by direct labeling ^[15,33,34]. A higher proliferation rate in HIV-infected subjects compared to uninfected individuals is not restricted to CD4 and CD8 T cells, but also observed in B cells, natural killer (NK) cells and macrophages ^[7,15,33]. Strong indirect support for the crucial role of immune activation in AIDS pathogenesis is provided by studies of SIV infections of natural hosts, in which high levels of virus replication are not sufficient to induce progression to AIDS in the absence of increased levels of immune activation ^[35-40].

This body of experimental evidence implicating a central role for immune activation in AIDS pathogenesis represents the backdrop for this article. Here we discuss the key questions that are central to this important issue in contemporary HIV/AIDS research.

Back to Top Article Outline

To what extent (if any) does immune activation reflect homeostatic pressure on the T-cell compartment?

There is wide consensus that during pathogenic HIV/SIV infection the level of T-cell proliferation and turnover is significantly increased when compared to that of healthy individuals. Conceptually, an increased T-cell turnover could reflect homeostatic proliferation in response to the cell losses induced by the virus or, alternatively, immune responses to antigenic stimulation and/or pro-inflammatory signals. These mechanisms are not mutually exclusive and in fact may be interrelated ^[41], but it should be kept in mind that while the above-mentioned ‘tap-and-drain’ model postulated an almost perfect quantitative balance between the rate of infected cell death on the one hand, and the rate of a compensatory/homeostatic CD4 T-cell proliferation on the other, several observations suggested a much more complex and indirect mechanistic and causal relations between CD4 T-cell killing by virus, immune activation, and T-cell turnover. First, CD8 T-cells, which are not directly targeted by the virus, also show increased rates of activation and proliferation ^[7,15,33]. Second, suppression of virus replication by antiretroviral therapy (ART) is followed by a rapid decline of proliferating CD4 T-cells at a time when the absolute number of these cells is still low ^[27]. Finally, the majority of CD4 T-cell death involves uninfected cells ^[42]. The currently prevailing view is that these cells die as a consequence of their previous activation, that their death is not directly responsible for the slow depletion of CD4 T-cells and that other consequences of chronic immune activation drive the pathogenic process leading to AIDS ^[9-17]. Grossman and colleagues also proposed that the heightened turnover of T cells during chronic HIV infection largely consists of overlapping bursts of proliferation and differentiation in response to T-cell receptor mediated stimuli and inflammation ^{[13,17,43,44]}. Direct support for the hypothesis that T-cell turnover is antigen driven has been provided by studies performed in SIV_mac239-infected rhesus macaques using extensive in vivo labeling of dividing cells with BrdU and tracing the kinetics of labeled T cells in blood and in lymphoid and nonlymphoid tissues ^[45,46].

Homeostatic pressure on the T-cell regenerative compartment likely occurs during pathogenic HIV/SIV infection, mainly as a consequence of the progressive depletion of naive and central memory T cells that are known to be subject to strict homeostatic regulation. Depletion of these cells, in turn, appears to be caused in large part by the chronic immune activation rather than the direct cytopathic effect of the virus. Furthermore, homeostatic proliferation (i.e., occurring in response to depletion) and classical immune activation-related proliferation (i.e., antigen-specific T-cell responses) are not necessarily distinct phenomena, but, rather, may overlap significantly. For instance, a scenario could be envisioned where a pro-inflammatory environment favors the activation of certain T-cell clones that may then become particularly prone to respond to homeostatic stimuli such as interleukin (IL)-7, IL-15 and others. Of note, linking ‘homeostatic proliferation’ to ‘immune activation’ in this way, within the framework of an immune activation oriented approach to the pathogenesis of HIV/SIV disease progression, bears no resemblance to a pathogenic model of HIV/SIV infection whereby CD4 T-cells are progressively depleted simply because their ‘homeostatic’ replication in response to viral killing collapses over time.

Another interesting question is how tissue-specific CD4 T-cell homeostasis (particularly in the mucosa associated lymphoid tissue, MALT) is maintained under normal circumstances and, in the context of HIV/SIV, whether and to what extent an increased homing of activated/memory CD4 T-cells in the MALT may compensate for the early loss of mucosal CD4CCR5 T cells. This point is important as the loss of mucosal CD4 T effector-memory (TEM) cells appears to be a critical determinant of progression to AIDS during both early and chronic phases of SIV infection of Indian rhesus macacques ^[45,46]. However, it is still unclear whether, in this model, the failure of reconstituting the mucosal CD4 TEM pool is primarily related to events occurring at the level of MALT (due to excessive virus-mediated cell destruction) as opposed to an upstream collapse of the CD4 central memory T (TCM) cell pool from which these CD4 TEM cells originate. A recent analysis of the dynamics of the input of CD4 T cells from the pool of lymph node-based TCM cells to that of MALT-based TEM cells during SIV infection supported the second view, although a defect in recruiting and/or retaining long-lived CD4 TEM cells in MALT due to the indirect effect of viral replication has also been implicated ^[46].

A better understanding of how CD4 T-cell homeostasis is regulated in the face of immune activation and how this regulation affects the physiologic events of CD4 T-cell activation, proliferation, and migration to effector tissues will help us elucidate the mechanisms of AIDS pathogenesis and hopefully pave the way to novel therapeutic approaches aimed directly at replenishing the CD4 T-cell pool in HIV-infected individuals.

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To what extent is immune activation a cause versus a consequence of the immune damage?

There is a broad consensus among investigators that, during pathogenic HIV/SIV infections, disease progression is closely associated with the level of immune activation. As discussed above, the majority of available data suggest that immune activation is most likely a cause of the damage to the immune system rather than being simply its consequence. Interestingly, immunologic studies in mice indicated that chronic immune activation may result in severe immune dysfunction and opportunistic infections even in absence of virus infection ^[47].

Naturally, immune activation that reflects specific responses to opportunistic infections (OI) can be considered a consequence of the immune dysfunction that was caused by the virus. These OI-specific immune responses, however, are a secondary and relatively late cause of immune activation, which is clearly established long before opportunistic infections occur. More complex is the relationship between immune activation and microbial translocation from a damaged intestinal lumen into systemic circulation. Recent work by Douek and his colleagues ^[48,49] suggests that the HIV/SIV-induced depletion of mucosal CD4 T cells results in the loss of mucosal integrity and thereby could trigger, or contribute to, the abnormal levels of chronic immune activation. It should be noted, however, that microbial translocation does not occur in SIV-infected sooty mangabeys (SM) and African green monkeys (AGM) despite a depletion of mucosal CD4 T cells that is comparable to that observed in pathogenic infections ^[49,52]. These latter observations indicate that factors other than the local depletion of CD4 T cells per se cause or contribute to the loss of mucosal integrity and microbial translocation associated with pathogenic HIV/SIV infection. Such additional factors might be related to the early establishment of pro-inflammatory tissue environments in human patients, but not in SM, or the depletion of non-CD4 T cells such as macrophages or dendritic cells during pathogenic infection. In any event, even assuming that all or most of the HIV-associated immune activation is caused by microbial translocation due to the loss of MALT CD4 T cells occurring during the first few weeks of infection, chronic immune activation remains the key to the ongoing systemic deletion of CD4 T cells, which is the best correlate to date of disease progression in humans.

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What causes immune activation in HIV infection?

While there is a broad consensus among investigators that immune activation plays an important role in AIDS pathogenesis, much debate remains as to what causes the HIV-associated immune activation. Many in the field now accept the idea that this phenomenon is multifactorial iature. We have compiled a list of potential factors that are most likely to contribute to the chronic, generalized immune activation observed during pathogenic HIV or SIV infection (Table 1). The first is the direct effect of HIV on T cells. HIV might directly influence immune activation through binding of the envelope protein gp120/160 to CD4 and/or CCR5, resulting in intracellular signaling ^[53-55]; or through the ability (or lack thereof) of HIVnef to down-modulate the expression of CD3-T cell receptor (TCR) in the infected cells ^[56]. The second factor capable of inducing systemic immune activation is the host immune response to HIV/SIV. This activation is likely to be initiated at the level of innate immunity – particularly involving plasmacytoid dendritic cells through Toll-like receptor (TLR) stimulation ^[57,58] – resulting in the activation of adaptive HIV-specific immune responses (humoral and cellular). The role of the virus-specific adaptive immune response (and, most notably, the HIV-specific cytotoxic T-cell response) is particularly complex due to its dual nature, i.e., beneficial as it may suppress virus replication, but harmful as it fuels chronic T-cell activation once the virus has escaped the immune response. Third, it was recently proposed that the HIV-associated immune activation is caused in part by translocation of microbial products from the intestinal lumen to the systemic circulation, where they can activate the immune system by binding to certain TLR (i.e., TLR-2, 4, 5, 6) ^[48,49]. This model postulates that microbial translocation (of which plasma levels of lipopolysaccahride is a reliable marker) occurs as a result of the depletion of intestinal lamina propria CD4 T cells and monocyte/macrophages through to direct cytopathic effect of the virus. It is also important to note that other pathogens, including but not limited to those causing OI during the later stages of disease, might also be playing roles in the HIV-associated immune activation ^[59-61]. For example, helminth infections may result in a more rapid progression to AIDS, possibly by augmenting the level of activation of the immune system ^[60]. A fourth potential factor is the non-antigen specific bystander activation of T and B lymphocytes caused by increased production of pro-inflammatory cytokines (e.g., tumor necrosis factor-α, IL-1, and others). This production, in turn, is also induced at the level of innate immune response to the HIV/SIV replication and is mediated by various types of accessory cells that are chronically activated. While the mechanisms of this ‘bystander’ activation are still relatively obscure, it is possible that they also involve the up-regulation of apoptosis related molecules (CD95, TRAIL, DR4/5) on the surface of T cells, thus making them prone to activation-induced cell death ^{[28-32,54,62]}. The last potential factor is the depletion and/or dysfunction of CD4 regulatory T cells (T_reg) that normally suppress immune activation via mechanisms involving direct cell-to-cell contact, production of cytokines, and inhibition of dendritic cell activity. The role of T_reg in HIV and SIV infection has been the subject of intense study over the past few years ^[63-76]. Conceivably, T_reg may play a dual role in HIV/SIV infection, i.e., protective if suppressing the chronic immune activation but harmful if attenuating effective T-cell responses. This dual role of T_reg, together with the fact that these cells appear to work in a tissue-specific manner, makes it difficult to interpret correlations between their number and functional state in blood samples and HIV disease progression.

Table 1
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Two still unanswered questions are: (i) why HIV infected individuals fail to effectively control the level of immune activation, as do natural host species infected with SIV, and (ii) why does the excessive activatioot resolve as it does in other chronic viral infections (e.g., hepatitis C virus, hepatitis B virus). While the comparison with these may not be altogether appropriate as these viruses do not preferentially infect immune system cells, the case of non-pathogenic SIV infection of African monkey species is particular intriguing as these infections are strikingly similar to pathogenic HIV/SIV infections in terms of the level of virus replication, target cell tropism, and ineffectiveness of antiviral immune responses ^[50,51,77].

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Why is the HIV-induced immune activation so disruptive to the immune system?

In considering this issue, it should be noted from the outset that although many lines of evidence indicate that chronic immune activation is a key determinant of immunodeficiency in HIV-infected individuals, the exact mechanisms by which this phenomenon induces CD4 T-cell depletion and disease progression are still largely unknown, and in fact may vary in different classes of patients. The possibilities discussed below are largely hypothetical.

Since HIV is known to replicate more efficiently in activated CD4 T lymphocytes ^[78], chronic immune activation is probably instrumental in sustaining viral replication by providing available targets for HIV replication. In this context, the preferential activation, infection and killing of HIV-specific CD4 T cells ^[79] is probably detrimental as it results in the loss of CD4 T-cell help, potentially contributing to the exhaustion/failure of CD8-mediated cytotoxic T lymphocytes responses to the virus. Another consequence of HIV-associated chronic immune activation that may have negative consequences in the long term is the expansion of activated ‘effector’ T (TE) cells of both CD4 and CD8 lineages ^[9,13,16]. The expansion of a pool of fast-replicating but short-lived CD4 TE cells may indirectly facilitate CD4 T-cell depletion. First, the expansion of CD4 TE cells may come at the expense of the naive and memory T-cell pools. A continuous drain from these pools could, in turn, result in a reduced capacity of the immune system to generate primary and anamnestic responses to antigens. Chronic immune activation may also result in the proliferative senescence of the T-cell pool, particularly at the level of CD4 TCM cells ^[46], thus supporting the interesting concept of AIDS as a disease characterized by a prematurely ageing immune system ^[80]. Second, expansion of activated TE cells may be accompanied by the production of pro-inflammatory and pro-apoptotic cytokines that complete the vicious cycle sustaining the generalized immune activation associated with pathogenic HIV/SIV infections. Third, the chronic pro-inflammatory environment has also multiple suppressive effects at different levels. It interferes with the function of several immune cell types, such as B cells, NK, γ δ T-cells, dendritic cells, and monocytes ^[81-86], and may impair the regenerative capacity of the immune system at the levels of bone marrow, thymus, and lymph nodes ^[87-90]. Interestingly, the increase in CD4 T cell counts that follows ART appears to be better correlated, at least in certain situations, with the favorable effect of ART on reducing immune activation and apoptosis rather than with its direct suppressive effect on HIV replication ^[91-94].

In summary, the hypothetical mechanisms by which T-cell immune activation causes disease progression in HIV-infected individuals can be grouped in three main classes: (i) stimulation of naive and memory CD4 T-cell activation, proliferation and differentiation, leading to increased CCR5 expression that renders these cells more susceptible to infection; (ii) alterations of long-term homeostasis of the naive and memory T-cell pools that lead to their gradual depletion and that interfere with the capacity of the host to effectively mount adaptive immune responses; (iii) induction of inflammation and fibrosis, likely destroying secondary lymphoid tissue niches required for the production and homeostasis of CD4 T cells.

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What experiments should be done to further test the immune activation hypothesis?

There is ample consensus among investigators that further experimentation – particularly in vivo studies conducted in the simian model of infection – is needed to ascertain and better characterize the pathogenic role of immune activation during HIV infection. Ideally, the best type of ‘experiment’ would involve treatment of SIV-infected macaques with drugs that either reduce or, alternatively, heighten the level of immune activation in vivo and then assess their effects on immune function and disease progression. Such experimental strategy should include treatment of SIV-infected macaques with TLR antagonists, chloroquine, or antibiotics. An additional interesting approach would be to determine whether artificially increasing the level of immune activation iatural SIV hosts such as SM and AGM (in which low immune activation is typically associated with a non-pathogenic infection) would result in signs of immunodeficiency. In this view, an interesting possibility is testing the ‘bacterial translocation’ hypothesis in SM and/or AGM by the administration of one or more bacterial TLR ligands to these animals during SIV infection. For studies aimed at modulating (i.e., increasing or decreasing) the HIV/SIV associated immune activation, the type, dose, and route of administration of the intervention agents, as well as the timing (acute versus chronic infection) are all important factors that require careful consideration in the design of these future experiments. More generally, it will be important to conduct studies aimed at determining which of the available models of pathogenic SIV infection in macaques (i.e., which virus, which species, etc.) demonstrates a degree of immune activation and disease progression that best resembles HIV infection in humans. Recent interesting comparative studies of Indian and Chinese rhesus macaques indicate that Indian rhesus tend to progress more rapidly to overt disease compared to Chinese rhesus ^[95]. The pattern of immune activation observed in Chinese macaques (particularly as assessed by the relative expansion of CD4CCR5 T cells) also suggests that infection of these animals may be more representative of HIV infection ^[96].

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Should we treat immune activation in HIV-infected patients? If so, when and how?

As mentioned above, a large set of data suggest that targeting the HIV-associated immune activation may represent a promising therapeutic strategy to be considered, in addition to ART, in the clinical management of HIV infection. However, the fact that the pathophysiologic mechanisms underlying this chronic activation are still poorly understood is a major obstacle to the implementation of a safe and effective immunosuppressive approach, especially when considering that, ultimately, HIV infection results in a state of immunodeficiency and that the wrong kind of immunosuppression might exacerbate this condition. The interventions should be carefully targeted, mechanism based and hypothesis driven, as preliminary studies have demonstrated that broad-spectrum immunosuppressive agents (such as cyclosporine and mycophenolate) are unlikely to provide the specificity that will enable the immune system to downregulate its hyperactivation and recover ^[97-101]. Novel and better ‘targeted’ immune interventions should be tested in short-term, proof-of-concept clinical trials conducted in small groups of well characterized patients treated during chronic infection (perhaps those defined, immunologically, as non-responders to ART or showing discordant response). As noted earlier, the line between ‘immune modulation’ and ‘immune reconstitution’ is not as clear-cut as was previously thought, and it is possible that the beneficial immunological effect of cytokines such as IL-2 and IL-7 may not only, or not primarily, lie in the improvement of CD4 T-cell homeostasis but also in reducing the prevailing level of T-cell activation and apoptosis. Finally, it is interesting to observe that ongoing clinical trials of CCR5 blockade in patients with dual-tropic viruses may allow us to assess whether blocking CCR5 signaling can reduce immune activation and improve the overall immune function, beyond the intended purpose of blocking virus entry and replication. In any immuno-modulatory intervention to be used in HIV-infected individuals, an important issue is how to best monitor changes in the existing level and pattern of immune activation. Unfortunately, none of the available cellular markers of T-cell activation or proliferation (HLA-DR, CD38, Ki67, loss of CD127, and others) seems to be able to consistently and robustly assess the level of the HIV-associated immune activation across all subsets of HIV-infected patients. It will be important to design studies in which multiple potential markers of immune activation are measured longitudinally in a sufficiently large cohort of HIV-infected individuals and the relative value of each of these markers, or of particular combinations, in predicting disease progression is assessed.

 

 

Introduction

The mechanisms by which HIV induces the immune dysfunction clinically defined as AIDS have been a subject of intense study since the discovery of the virus in the early 1980s. Initial virological analysis demonstrated low levels of virus replication in infected patients, suggesting that the virus alone was incapable of inducing AIDS and that additional factors must also play a role in determining the HIV-associated immunologic dysfunction. This concept has recently been emphasized from a statistical perspective by the observation that while the level of HIV replication is significantly correlated with the risk of disease progression ^[1], this parameter only predicts a minor part of the variation in the rate of progression among infected patients ^[2]. In the mid 1990s, improvements in the techniques available to detect HIV demonstrated that virus replication was active throughout the course of the disease ^[3,4]. In addition, the observation that inhibition of viral replication with antiretroviral drugs substantially attenuates disease progression established very clearly that virus replication is responsible for pathogenicity. What remains poorly defined, however, is the mechanistic linkage between virus replication and the onset of AIDS.

A model of CD4 T-cell depletion based entirely on direct virus infection and killing of these cells was put forward in the mid 1990s ^[5,6]. This so-called ‘tap-and-drain’ model proposed that progression to AIDS in HIV-infected individuals resulted from a failure of the immune system’s homeostatic response to keep up with a high rate of loss of CD4 T cells ^[5,6]. The model offered an explanation for the rapid increase of CD4 T-cell counts following inhibition of virus replication by antiretroviral therapy. However, this model and its later versions ^[7,8] were challenged on theoretical and experimental grounds as they did not appear to grasp the complexity of T-cell dynamics in response to ongoing viral replication and painted a simplistic picture of AIDS pathogenesis ^[9-17].

The idea that chronic immune activation plays a major role in AIDS pathogenesis was first put forward by Ascher and Sheppard ^[18] and, in parallel – but from a rather different perspective – by Grossman and colleagues in the late 1980s/early 1990s ^[19,20]. Shortly thereafter, Giorgi and colleagues published a series of clinical studies supporting the concept that an excessive/aberrant immune activation is a fundamental driving force for the HIV-associated immune dysfunction. These studies identified the level of CD8 T-cell activation, as determined by CD38 and HLA-DR expression, as a better correlate of disease progression than viral load ^[21-24]. While exact characterization of the HIV-associated chronic immune activation remains incomplete, an activation/dysfunction phenotype is apparent for many different immune cell types in HIV infection. With regard to T-cells, the assessment of immune activation can be made through: (i) high frequency of T cells expressing markers of activation and proliferation ^[25-27]; (ii) high levels of activation-induced apoptosis of uninfected T cells ^[28-32]; (iii) high levels of T-cell proliferation as measured by direct labeling ^[15,33,34]. A higher proliferation rate in HIV-infected subjects compared to uninfected individuals is not restricted to CD4 and CD8 T cells, but also observed in B cells, natural killer (NK) cells and macrophages ^[7,15,33]. Strong indirect support for the crucial role of immune activation in AIDS pathogenesis is provided by studies of SIV infections of natural hosts, in which high levels of virus replication are not sufficient to induce progression to AIDS in the absence of increased levels of immune activation ^[35-40].

This body of experimental evidence implicating a central role for immune activation in AIDS pathogenesis represents the backdrop for this article. Here we discuss the key questions that are central to this important issue in contemporary HIV/AIDS research.

Back to Top Article Outline

To what extent (if any) does immune activation reflect homeostatic pressure on the T-cell compartment?

There is wide consensus that during pathogenic HIV/SIV infection the level of T-cell proliferation and turnover is significantly increased when compared to that of healthy individuals. Conceptually, an increased T-cell turnover could reflect homeostatic proliferation in response to the cell losses induced by the virus or, alternatively, immune responses to antigenic stimulation and/or pro-inflammatory signals. These mechanisms are not mutually exclusive and in fact may be interrelated ^[41], but it should be kept in mind that while the above-mentioned ‘tap-and-drain’ model postulated an almost perfect quantitative balance between the rate of infected cell death on the one hand, and the rate of a compensatory/homeostatic CD4 T-cell proliferation on the other, several observations suggested a much more complex and indirect mechanistic and causal relations between CD4 T-cell killing by virus, immune activation, and T-cell turnover. First, CD8 T-cells, which are not directly targeted by the virus, also show increased rates of activation and proliferation ^[7,15,33]. Second, suppression of virus replication by antiretroviral therapy (ART) is followed by a rapid decline of proliferating CD4 T-cells at a time when the absolute number of these cells is still low ^[27]. Finally, the majority of CD4 T-cell death involves uninfected cells ^[42]. The currently prevailing view is that these cells die as a consequence of their previous activation, that their death is not directly responsible for the slow depletion of CD4 T-cells and that other consequences of chronic immune activation drive the pathogenic process leading to AIDS ^[9-17]. Grossman and colleagues also proposed that the heightened turnover of T cells during chronic HIV infection largely consists of overlapping bursts of proliferation and differentiation in response to T-cell receptor mediated stimuli and inflammation ^{[13,17,43,44]}. Direct support for the hypothesis that T-cell turnover is antigen driven has been provided by studies performed in SIV_mac239-infected rhesus macaques using extensive in vivo labeling of dividing cells with BrdU and tracing the kinetics of labeled T cells in blood and in lymphoid and nonlymphoid tissues ^[45,46].

Homeostatic pressure on the T-cell regenerative compartment likely occurs during pathogenic HIV/SIV infection, mainly as a consequence of the progressive depletion of naive and central memory T cells that are known to be subject to strict homeostatic regulation. Depletion of these cells, in turn, appears to be caused in large part by the chronic immune activation rather than the direct cytopathic effect of the virus. Furthermore, homeostatic proliferation (i.e., occurring in response to depletion) and classical immune activation-related proliferation (i.e., antigen-specific T-cell responses) are not necessarily distinct phenomena, but, rather, may overlap significantly. For instance, a scenario could be envisioned where a pro-inflammatory environment favors the activation of certain T-cell clones that may then become particularly prone to respond to homeostatic stimuli such as interleukin (IL)-7, IL-15 and others. Of note, linking ‘homeostatic proliferation’ to ‘immune activation’ in this way, within the framework of an immune activation oriented approach to the pathogenesis of HIV/SIV disease progression, bears no resemblance to a pathogenic model of HIV/SIV infection whereby CD4 T-cells are progressively depleted simply because their ‘homeostatic’ replication in response to viral killing collapses over time.

Another interesting question is how tissue-specific CD4 T-cell homeostasis (particularly in the mucosa associated lymphoid tissue, MALT) is maintained under normal circumstances and, in the context of HIV/SIV, whether and to what extent an increased homing of activated/memory CD4 T-cells in the MALT may compensate for the early loss of mucosal CD4CCR5 T cells. This point is important as the loss of mucosal CD4 T effector-memory (TEM) cells appears to be a critical determinant of progression to AIDS during both early and chronic phases of SIV infection of Indian rhesus macacques ^[45,46]. However, it is still unclear whether, in this model, the failure of reconstituting the mucosal CD4 TEM pool is primarily related to events occurring at the level of MALT (due to excessive virus-mediated cell destruction) as opposed to an upstream collapse of the CD4 central memory T (TCM) cell pool from which these CD4 TEM cells originate. A recent analysis of the dynamics of the input of CD4 T cells from the pool of lymph node-based TCM cells to that of MALT-based TEM cells during SIV infection supported the second view, although a defect in recruiting and/or retaining long-lived CD4 TEM cells in MALT due to the indirect effect of viral replication has also been implicated ^[46].

A better understanding of how CD4 T-cell homeostasis is regulated in the face of immune activation and how this regulation affects the physiologic events of CD4 T-cell activation, proliferation, and migration to effector tissues will help us elucidate the mechanisms of AIDS pathogenesis and hopefully pave the way to novel therapeutic approaches aimed directly at replenishing the CD4 T-cell pool in HIV-infected individuals.

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To what extent is immune activation a cause versus a consequence of the immune damage?

There is a broad consensus among investigators that, during pathogenic HIV/SIV infections, disease progression is closely associated with the level of immune activation. As discussed above, the majority of available data suggest that immune activation is most likely a cause of the damage to the immune system rather than being simply its consequence. Interestingly, immunologic studies in mice indicated that chronic immune activation may result in severe immune dysfunction and opportunistic infections even in absence of virus infection ^[47].

Naturally, immune activation that reflects specific responses to opportunistic infections (OI) can be considered a consequence of the immune dysfunction that was caused by the virus. These OI-specific immune responses, however, are a secondary and relatively late cause of immune activation, which is clearly established long before opportunistic infections occur. More complex is the relationship between immune activation and microbial translocation from a damaged intestinal lumen into systemic circulation. Recent work by Douek and his colleagues ^[48,49] suggests that the HIV/SIV-induced depletion of mucosal CD4 T cells results in the loss of mucosal integrity and thereby could trigger, or contribute to, the abnormal levels of chronic immune activation. It should be noted, however, that microbial translocation does not occur in SIV-infected sooty mangabeys (SM) and African green monkeys (AGM) despite a depletion of mucosal CD4 T cells that is comparable to that observed in pathogenic infections ^[49,52]. These latter observations indicate that factors other than the local depletion of CD4 T cells per se cause or contribute to the loss of mucosal integrity and microbial translocation associated with pathogenic HIV/SIV infection. Such additional factors might be related to the early establishment of pro-inflammatory tissue environments in human patients, but not in SM, or the depletion of non-CD4 T cells such as macrophages or dendritic cells during pathogenic infection. In any event, even assuming that all or most of the HIV-associated immune activation is caused by microbial translocation due to the loss of MALT CD4 T cells occurring during the first few weeks of infection, chronic immune activation remains the key to the ongoing systemic deletion of CD4 T cells, which is the best correlate to date of disease progression in humans.

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What causes immune activation in HIV infection?

While there is a broad consensus among investigators that immune activation plays an important role in AIDS pathogenesis, much debate remains as to what causes the HIV-associated immune activation. Many in the field now accept the idea that this phenomenon is multifactorial iature. We have compiled a list of potential factors that are most likely to contribute to the chronic, generalized immune activation observed during pathogenic HIV or SIV infection (Table 1). The first is the direct effect of HIV on T cells. HIV might directly influence immune activation through binding of the envelope protein gp120/160 to CD4 and/or CCR5, resulting in intracellular signaling ^[53-55]; or through the ability (or lack thereof) of HIVnef to down-modulate the expression of CD3-T cell receptor (TCR) in the infected cells ^[56]. The second factor capable of inducing systemic immune activation is the host immune response to HIV/SIV. This activation is likely to be initiated at the level of innate immunity – particularly involving plasmacytoid dendritic cells through Toll-like receptor (TLR) stimulation ^[57,58] – resulting in the activation of adaptive HIV-specific immune responses (humoral and cellular). The role of the virus-specific adaptive immune response (and, most notably, the HIV-specific cytotoxic T-cell response) is particularly complex due to its dual nature, i.e., beneficial as it may suppress virus replication, but harmful as it fuels chronic T-cell activation once the virus has escaped the immune response. Third, it was recently proposed that the HIV-associated immune activation is caused in part by translocation of microbial products from the intestinal lumen to the systemic circulation, where they can activate the immune system by binding to certain TLR (i.e., TLR-2, 4, 5, 6) ^[48,49]. This model postulates that microbial translocation (of which plasma levels of lipopolysaccahride is a reliable marker) occurs as a result of the depletion of intestinal lamina propria CD4 T cells and monocyte/macrophages through to direct cytopathic effect of the virus. It is also important to note that other pathogens, including but not limited to those causing OI during the later stages of disease, might also be playing roles in the HIV-associated immune activation ^[59-61]. For example, helminth infections may result in a more rapid progression to AIDS, possibly by augmenting the level of activation of the immune system ^[60]. A fourth potential factor is the non-antigen specific bystander activation of T and B lymphocytes caused by increased production of pro-inflammatory cytokines (e.g., tumor necrosis factor-α, IL-1, and others). This production, in turn, is also induced at the level of innate immune response to the HIV/SIV replication and is mediated by various types of accessory cells that are chronically activated. While the mechanisms of this ‘bystander’ activation are still relatively obscure, it is possible that they also involve the up-regulation of apoptosis related molecules (CD95, TRAIL, DR4/5) on the surface of T cells, thus making them prone to activation-induced cell death ^{[28-32,54,62]}. The last potential factor is the depletion and/or dysfunction of CD4 regulatory T cells (T_reg) that normally suppress immune activation via mechanisms involving direct cell-to-cell contact, production of cytokines, and inhibition of dendritic cell activity. The role of T_reg in HIV and SIV infection has been the subject of intense study over the past few years ^[63-76]. Conceivably, T_reg may play a dual role in HIV/SIV infection, i.e., protective if suppressing the chronic immune activation but harmful if attenuating effective T-cell responses. This dual role of T_reg, together with the fact that these cells appear to work in a tissue-specific manner, makes it difficult to interpret correlations between their number and functional state in blood samples and HIV disease progression.

Table 1
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Two still unanswered questions are: (i) why HIV infected individuals fail to effectively control the level of immune activation, as do natural host species infected with SIV, and (ii) why does the excessive activatioot resolve as it does in other chronic viral infections (e.g., hepatitis C virus, hepatitis B virus). While the comparison with these may not be altogether appropriate as these viruses do not preferentially infect immune system cells, the case of non-pathogenic SIV infection of African monkey species is particular intriguing as these infections are strikingly similar to pathogenic HIV/SIV infections in terms of the level of virus replication, target cell tropism, and ineffectiveness of antiviral immune responses ^[50,51,77].

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Why is the HIV-induced immune activation so disruptive to the immune system?

In considering this issue, it should be noted from the outset that although many lines of evidence indicate that chronic immune activation is a key determinant of immunodeficiency in HIV-infected individuals, the exact mechanisms by which this phenomenon induces CD4 T-cell depletion and disease progression are still largely unknown, and in fact may vary in different classes of patients. The possibilities discussed below are largely hypothetical.

Since HIV is known to replicate more efficiently in activated CD4 T lymphocytes ^[78], chronic immune activation is probably instrumental in sustaining viral replication by providing available targets for HIV replication. In this context, the preferential activation, infection and killing of HIV-specific CD4 T cells ^[79] is probably detrimental as it results in the loss of CD4 T-cell help, potentially contributing to the exhaustion/failure of CD8-mediated cytotoxic T lymphocytes responses to the virus. Another consequence of HIV-associated chronic immune activation that may have negative consequences in the long term is the expansion of activated ‘effector’ T (TE) cells of both CD4 and CD8 lineages ^[9,13,16]. The expansion of a pool of fast-replicating but short-lived CD4 TE cells may indirectly facilitate CD4 T-cell depletion. First, the expansion of CD4 TE cells may come at the expense of the naive and memory T-cell pools. A continuous drain from these pools could, in turn, result in a reduced capacity of the immune system to generate primary and anamnestic responses to antigens. Chronic immune activation may also result in the proliferative senescence of the T-cell pool, particularly at the level of CD4 TCM cells ^[46], thus supporting the interesting concept of AIDS as a disease characterized by a prematurely ageing immune system ^[80]. Second, expansion of activated TE cells may be accompanied by the production of pro-inflammatory and pro-apoptotic cytokines that complete the vicious cycle sustaining the generalized immune activation associated with pathogenic HIV/SIV infections. Third, the chronic pro-inflammatory environment has also multiple suppressive effects at different levels. It interferes with the function of several immune cell types, such as B cells, NK, γ δ T-cells, dendritic cells, and monocytes ^[81-86], and may impair the regenerative capacity of the immune system at the levels of bone marrow, thymus, and lymph nodes ^[87-90]. Interestingly, the increase in CD4 T cell counts that follows ART appears to be better correlated, at least in certain situations, with the favorable effect of ART on reducing immune activation and apoptosis rather than with its direct suppressive effect on HIV replication ^[91-94].

In summary, the hypothetical mechanisms by which T-cell immune activation causes disease progression in HIV-infected individuals can be grouped in three main classes: (i) stimulation of naive and memory CD4 T-cell activation, proliferation and differentiation, leading to increased CCR5 expression that renders these cells more susceptible to infection; (ii) alterations of long-term homeostasis of the naive and memory T-cell pools that lead to their gradual depletion and that interfere with the capacity of the host to effectively mount adaptive immune responses; (iii) induction of inflammation and fibrosis, likely destroying secondary lymphoid tissue niches required for the production and homeostasis of CD4 T cells.

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What experiments should be done to further test the immune activation hypothesis?

There is ample consensus among investigators that further experimentation – particularly in vivo studies conducted in the simian model of infection – is needed to ascertain and better characterize the pathogenic role of immune activation during HIV infection. Ideally, the best type of ‘experiment’ would involve treatment of SIV-infected macaques with drugs that either reduce or, alternatively, heighten the level of immune activation in vivo and then assess their effects on immune function and disease progression. Such experimental strategy should include treatment of SIV-infected macaques with TLR antagonists, chloroquine, or antibiotics. An additional interesting approach would be to determine whether artificially increasing the level of immune activation iatural SIV hosts such as SM and AGM (in which low immune activation is typically associated with a non-pathogenic infection) would result in signs of immunodeficiency. In this view, an interesting possibility is testing the ‘bacterial translocation’ hypothesis in SM and/or AGM by the administration of one or more bacterial TLR ligands to these animals during SIV infection. For studies aimed at modulating (i.e., increasing or decreasing) the HIV/SIV associated immune activation, the type, dose, and route of administration of the intervention agents, as well as the timing (acute versus chronic infection) are all important factors that require careful consideration in the design of these future experiments. More generally, it will be important to conduct studies aimed at determining which of the available models of pathogenic SIV infection in macaques (i.e., which virus, which species, etc.) demonstrates a degree of immune activation and disease progression that best resembles HIV infection in humans. Recent interesting comparative studies of Indian and Chinese rhesus macaques indicate that Indian rhesus tend to progress more rapidly to overt disease compared to Chinese rhesus ^[95]. The pattern of immune activation observed in Chinese macaques (particularly as assessed by the relative expansion of CD4CCR5 T cells) also suggests that infection of these animals may be more representative of HIV infection ^[96].

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Should we treat immune activation in HIV-infected patients? If so, when and how?

As mentioned above, a large set of data suggest that targeting the HIV-associated immune activation may represent a promising therapeutic strategy to be considered, in addition to ART, in the clinical management of HIV infection. However, the fact that the pathophysiologic mechanisms underlying this chronic activation are still poorly understood is a major obstacle to the implementation of a safe and effective immunosuppressive approach, especially when considering that, ultimately, HIV infection results in a state of immunodeficiency and that the wrong kind of immunosuppression might exacerbate this condition. The interventions should be carefully targeted, mechanism based and hypothesis driven, as preliminary studies have demonstrated that broad-spectrum immunosuppressive agents (such as cyclosporine and mycophenolate) are unlikely to provide the specificity that will enable the immune system to downregulate its hyperactivation and recover ^[97-101]. Novel and better ‘targeted’ immune interventions should be tested in short-term, proof-of-concept clinical trials conducted in small groups of well characterized patients treated during chronic infection (perhaps those defined, immunologically, as non-responders to ART or showing discordant response). As noted earlier, the line between ‘immune modulation’ and ‘immune reconstitution’ is not as clear-cut as was previously thought, and it is possible that the beneficial immunological effect of cytokines such as IL-2 and IL-7 may not only, or not primarily, lie in the improvement of CD4 T-cell homeostasis but also in reducing the prevailing level of T-cell activation and apoptosis. Finally, it is interesting to observe that ongoing clinical trials of CCR5 blockade in patients with dual-tropic viruses may allow us to assess whether blocking CCR5 signaling can reduce immune activation and improve the overall immune function, beyond the intended purpose of blocking virus entry and replication. In any immuno-modulatory intervention to be used in HIV-infected individuals, an important issue is how to best monitor changes in the existing level and pattern of immune activation. Unfortunately, none of the available cellular markers of T-cell activation or proliferation (HLA-DR, CD38, Ki67, loss of CD127, and others) seems to be able to consistently and robustly assess the level of the HIV-associated immune activation across all subsets of HIV-infected patients. It will be important to design studies in which multiple potential markers of immune activation are measured longitudinally in a sufficiently large cohort of HIV-infected individuals and the relative value of each of these markers, or of particular combinations, in predicting disease progression is assessed.

 

DIAGNOSIS

Many HIV-positive people are unaware that they are infected with the virus.^[62] For example, in 2001 less than 1% of the sexually active urban population in Africa had been tested, and this proportion is even lower in rural populations.^[62] Furthermore, in 2001 only 0.5% of pregnant women attending urban health facilities are counselled, tested or receive their test results.^[62] Again, this proportion is even lower in rural health facilities.^[62] Since donors may therefore be unaware of their infection, donor blood and blood products used in medicine and medical research are routinely screened for HIV.^[63]

HIV-1 testing is initially by an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to HIV-1. Specimens with a nonreactive result from the initial ELISA are considered HIV-negative unless new exposure to an infected partner or partner of unknown HIV status has occurred. Specimens with a reactive ELISA result are retested in duplicate.^[64] If the result of either duplicate test is reactive, the specimen is reported as repeatedly reactive and undergoes confirmatory testing with a more specific supplemental test (e.g., Western blot or, less commonly, an immunofluorescence assay (IFA)). Only specimens that are repeatedly reactive by ELISA and positive by IFA or reactive by Western blot are considered HIV-positive and indicative of HIV infection. Specimens that are repeatedly ELISA-reactive occasionally provide an indeterminate Western blot result, which may be either an incomplete antibody response to HIV in an infected person or nonspecific reactions in an uninfected person.^[65]

Although IFA can be used to confirm infection in these ambiguous cases, this assay is not widely used. In general, a second specimen should be collected more than a month later and retested for persons with indeterminate Western blot results. Although much less commonly available, nucleic acid testing (e.g., viral RNA or proviral DNA amplification method) can also help diagnosis in certain situations.^[64] In addition, a few tested specimens might provide inconclusive results because of a low quantity specimen. In these situations, a second specimen is collected and tested for HIV infection.

Modern HIV testing is extremely accurate. A single screening test is correct more than 99% of the time.^{[66][needs update]} The chance of a false-positive result in standard two-step testing protocol is estimated to be about 1 in 250,000 in a low risk population.^[66] Testing post exposure is recommended initially and at six weeks, three months, and six months.^[67] 

 

http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Hiv-timecourse_copy.svg/729px-Hiv-timecourse_copy.svg.png

http://www.google.com.ua/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&sqi=2&ved=0CDgQFjAA&url=http%3A%2F%2Fwww.pitt.edu%2F~super7%2F19011-20001%2F19601.pdf&ei=mZlAUfWpGM3Aswam2IDYDQ&usg=AFQjCNGiGUCIoWeTN79XK41zTtUW534Trw&sig2=5Z58V61LJds6rOLMLyRWCA&bvm=bv.43287494,d.Yms&cad=rj

 

Although IFA can be used to confirm infection in these ambiguous cases, this assay is not widely used. In general, a second specimen should be collected more than a month later and retested for persons with indeterminate Western blot results. Although much less commonly available, nucleic acid testing (e.g., viral RNA or proviral DNA amplification method) can also help diagnosis in certain situations.^[64] In addition, a few tested specimens might provide inconclusive results because of a low quantity specimen. In these situations, a second specimen is collected and tested for HIV infection.

Modern HIV testing is extremely accurate. A single screening test is correct more than 99% of the time.^{[66][needs update]} The chance of a false-positive result in standard two-step testing protocol is estimated to be about 1 in 250,000 in a low risk population.^[66] Testing post exposure is recommended initially and at six weeks, three months, and six months.^[67] 

 

Acquired Immunodeficiency Syndrome Treatment and prevention

Strategies for the  treatment and prevention of the HIV infection have been focused in a large part of inhibiting different stages of the HIV replication cycle. The most successful treatment modality makes use of the drug AZT,  3′-azido-3′-deoxythymidine,  also known as zidovudine or Retrovir.  AZT,  first synthesized in 1964,  is a potent inhibitor of HIV replication in vitro and specifically inhibits reverse transcription.  The antiretroviral activity of AZT is a result of a preferential interaction of the 5′-triphosphate of AZT with the viral reverse transcriptase,  inhibiting viral reverse transcriptase activity about 100 times more efficiently than do cellular DNA polymerases.  After clinical trials in 1986,  AZT was licensed in the United States during 1987 for the treatment of patients with symptomatic HIV infections.  AZT is not without sometimes serious side effects, the major ones being macrocytic anemia and granulocytopenia.  However,  the most severe problem is that HIV variants resistant to AZT invariably arise in treated patients.  Thus,  although the initial effects of treatment often are dramatic,  with diminishing viral replication and some-times increasing T4-cell levels,  the effects are transient. Several other reverse transcriptase inhibitors have been developed,  but resistance is a problem with these drugs as well.  Most recently,  novel drugs have been developed that specifically inhibit another enzyme,  the viral protease. Some of these compounds are undergoing clinical trials, but it already has been documented that the rapid development of resistant virus variants is going to limit the usefulness of these drugs.

Initial infection with HIV is sometimes accompanied by mild flu-like symptoms such as fever, sore throat, and fatigue. Thereafter, HIV-infectcd individuals may be symptom free for a long period (5 to 7 years on the average), although Th cell counts progressively declines. Ultimately, individuals with AIDS exhibit i mm uno suppress ion. As a result, they are subject to infection by numerous opportunistic pathogens, as well as true pathogens, and to development of several forms of cancer (Table 1). Generally one infection follows another in people with this disease until death occurs. In 1993, the case definition for AIDS included individuals who: (1) tested HIV positive (ELISA assay for HIV p25 viral core antigen), (2) had less than 200 CD4 T lymphocytes/mm³ of blood—instead of 800-1300 CD4 T lymphocytes/mm³ in healthy individuals, or (3) greater than 200 CD4 T lymphocytes/mm³ of blood and opportunistic infection or cancer.

In spite of global efforts,  an effective vaccine for HIV has not been developed.  Most of the potential vaccines that have been tested have consisted of different forms of the HIV envelope protein.  It has been shown in bothh uman vaccine trials and animal experiments that these vaccines are capable of inducing good immune responses, including the induction of neutralizing antibodies.  However,  these antibodies are only able to protect against an infection with a virus that contains the same or a similar envelope protein.  This presents a serious problem,  because many different virus variants with vastly different envelope proteins are circulating in the human population.  In fact,  it has been shown that many different viruses may exist simultaneously within the same patient,  and that the predominant variant changes from time to time. This is likely due to the selection of viruses that are not neutralized by the prevalent neutralizing antibodies, which helps to explain why the virus can continue to replicate in infected individuals in spite of a generally good initial immune response.  Vaccines based on other viral components are under development,  and preparations containing a mixture of several different envelope proteins also are under consideration.

A Closer Look

In the spring of 1981,  the first cases of the disease that soon became known as AIDS (Acquired Immune Deficiency Syndrome) started to appear at hospitals in New York City,  San Francisco,  and Los Angeles.  The patients all presented with severe opportunistic infections,  and several of them had a form of pneumonia caused by Pneumocystic carinii that had previously been extremely rare in the United States.  Almost all of the patients were gay men.  At the same time,  several young gay men were diagnosed with a strange skin condition.  The lesions were soon recognized to be consistent with Kaposi’s sarcoma,  a rare form of cancer that causes characteristic skin lesions.  What was particularly puzzling was that this disease had previously been diagnosed almost exclusively in elderly Jewish and Italian men.  In addition,  the skin cancer in the young men seemed much more aggressive than the usual cases of Kaposi’s sarcoma.

Table

Principal Opportunistic Pathogens in AIDS Patients

 

 

The underlying cause of all these problems was soon recognized to be a severe defect in the patients’ immune systems.  The low numbers of CD4 helper T cells in their blood were particularly striking.  Up to this time,  severe immunodeficiencies had been relatively rare and were usually associated with genetic defects or aggressive immunosuppressive treatments.

As the number of cases started to increase,  it became clear that an epidemic was emerging among gay men. Soon,  similar cases were reported from several countries in Europe.  Although an infectious agent was suspected almost from the beginning,  it was also possible that the disease could be caused by an environmental agent.  Potential candidates were some of the drugs that were commonly used in the gay community.  However, many virologists started to suspect that the disease might be caused by a virus,  and by 1982,  the hunt for the potential culprit was on.

Tissues and blood from patients with the disease were examined for every possible virus,  and for a while, the disease was believed to be caused by a herpesvirus or an adenovirus.  In the spring of 1983,  however,  a group at the French Pasteur Institute led by Dr.  Luc Montagnier,  reported that they had isolated what seemed to be a novel retrovirus from several patients with AIDS.  Their findings were later confirmed by isolations of similar viruses by Dr.  Robert Gallo’s group at the National Institutes of Health (NIH) and by Dr. Jay Levy’s group in San Francisco.

By the time the novel retrovirus was discovered,  it had become obvious that AIDS was transmitted in three different ways: by sexual transmission,  through contaminated blood,  and from an infected mother to her child.  Indications of these routes of transmission included the findings that an increasing number of cases involved intravenous drug users and children born to mothers who were intravenous drug users. Several cases of the disease had already been reported in recipients of blood transfusions,  in hemophiliacs who had received blood concentrates,  and in children of each of these two groups.  It thus became imperative to quickly develop a blood test for the virus.  Through collaborative efforts between the groups that had first isolated the virus (originally named LAV by the French group,  ARV by the California group,  and HTLV-III by the NIH group),  such a test soon became available. It thus became possible to test every batch of blood and blood concentrate for the presence of the virus, which was now called human immunodeficiency virus, or HIV.  Testing of blood supply and blood products soon became mandatory in most countries of the world.

As soon as the blood test became available,  researchers saw that the picture of AIDS was even more chilling than originally suspected.  It was soon established that the virus was present not only in the United States and Europe,  but also in several other areas of the world. The picture was especially grim in Africa,  which is now believed to be where the virus originated.  Random testing showed that the epidemic was rampant in several different African countries,  with some cities showing overall seroprevalence levels of approximately 10% to 15%.  This testing also showed that AIDS was already a major killer in Africa—a fact that had been largely unrecognized because of the multitude of other medical problems and deaths caused by malnutrition and a low standard of living.

The blood test also showed that 30% to 40% of hemophiliacs were infected and that many more recipients of blood transfusions were infected than had originally been suspected.  Many of these individuals showed little or no symptoms of AIDS.  Because the exact date on which these patients had been infected was usually possible to pinpoint,  researchers realized that many of these patients had already lived several years with the virus.  We now know that the latency period between HIV infection and the development of AIDS can be 10 or more years.  Some long-term survivors have shown little or no signs of a declining immune system 10 to 15 years after they were infected.  About 5% of patients seem to belong in this group.

 

 

 

Viral Oncogenes and Cellular Transformation

The realization that acute transforming rctroviruses contain oncogenes that are both necessary and sufficient For the transformation of cells in culture and for the induction of animal tumors led investigators to initiate an analysis of the proteins encoded by these oncogenes. Between 25 and 30 viral oncogenes have been identified and are designated bv three letter acronyms. In most cases, the viral oncogene products have been identified Table 44 1 summarizes our knowledge of representative members of the different oncogene families. The discovery that viral oncogenes were acquired from normal host
genes (proto oncogenes) supported the notion that viral oncoproteins would likely be related structurally and functionally to important cellular host proteins and enzymes. In many cases, DNA and protein sequence analysis has permitted the identification of the cellular homologue ot the viral oncogene, verifying this notion. For example, the oncoprotem encoded by the simian sarcoma virus, v-sis, is structurally identical to a growth factor secreted by platelets, platelet derived growth factor (PDGF), whereas the oncoprotein encoded bv the avian erythroblastosis virus erb B gene is structurally identical to a portion of the receptor for epidermal growth factor (EGF). These and other examples are summarized in Table.

Table

Viral Oncogenes and Cellular Homologues of Known Functionr

 

GTP, guanosine triphosphate; GDP, guanosine diphosphate

 

Understanding of the biochemical functions mediated by oncoproteins has provided important insights into the types of cellular perturbations that leid to cellular transformation. The properties of some of these proteins are summarized here.

 

 

Oncogene Families

Viral oncoproteins can function in either the nucleus or the cytoplasm. Sequence analysis and intracellular location, as well as functional activity, have permitted the general classification of these proteins into families (Table).

Viral Oncogenes and Their Products

 

AVS, avian sarcoma virus; FeSV, feline sarcoma virus; M1L, mouse leukemia virus;  AEV, avian erythroblastosis virus;  SSV, simian sarcoma virus; AMV avian myeloblastosis virus; REV,  reticuloendotheliosis virus; MSV mouse sarcoma virus; RSV, Rous sarcoma virus;  ST, Synder-Theilin; GR, Gardner-Rasheed; SM, Susan McDonough;FBR, Finkel-Biskis- Reilly; GTP, guanosine triphosphate; GDP,  guanosine diphosphate; PDGF, platelet-derived growth factor.

* Identifed by DNA transfection of mouse cells. ** Not found in a retrovirus. *** Function unknown.

 

The largest family is made up of the tyrosine kinase oncogenes (representative members being src, abl, fps, yes, fgr, fms, ros, and erb B). The proteins encoded by these oncogenes are found associated with cellular membranes and exhibit protein tyrosine kinase activity (ie, the transfer of phosphate from adenosine triphosphate to tyrosine residues in the acceptor protein). The members of this family can be subdivided further into membrane spanning tyrosine kinases that resemble growth factor receptors (v-fms, v-erb B) and tyrosine kinases that associate with the inner side of the plasma membrane (v-src, v-yes, v fps). The significance of this distinction is discussed later. A closely related family of oncogenes (mos, raf, mil, and rel) has been shown to have partial sequence similarity with the tyrosine kinase family of oncogenes. Several members of this family exhibit protein serine/threonine
kinase activity. Also in the cytoplasm is the ras family of oncogenes (Ha-ras, K-ras, and N-ras). Members of this highly conserved family of oncoproteins are characterized by their ability to bind guanine nucleotides and hydrolyze guanosine triphosphate (GTP) (guanosine diphosphate [GDP]/GTP binding/GTPase activity). The nuclear oncogene family includes erb A, jun, fos, myc, myb, ski, and two DNA tumour virus gene products, adeno virus El A and SV40/polyoma T antigen. Nuclear oncogenes function in a variety of ways to alter the pattern of gene expression. For example, DNA sequence analysis has shown that erb A is an altered form of the thyroxine receptor and appears to interact with a unique receptor-binding site. Similarly, jun and fos appear to function as “transcription” factors by binding directly to DNA and promoting gene expression.

THE TYROSINE KINASE FAMILY

Perhaps the best studied of all the viral oncoproteins is the src protein of the acute transforming virus, Rous sarcoma virus (RSV). RSV induces sarcomas in chickens at the site of injection and transforms a variety of avian and rodent cells in cell culture. All cells transformed by RSV produce a 60 kilodalton protein that can be identified in whole cell extracts by immunoprecipitation with antisera fromrabbits or mice bearing RSV-induced tumours. The most striking feature of the src protein, designated pp60^src, is that it is a protein tyrosine kinase capable of phosphorylation of either itself (autophosphorylation) or certain other proteins on tyrosine residues. This unusual kinase activity of pp60^<a is particularly interesting in light of the observation that iormal cells, only about 0.01% of all phosphoamino acids in proteins are phosphotyrosine, most being phosphoserine (99%) and phosphothreonine (0.09%). In cells transformed with RSV, the level of tyrosine phosphorylation in cellular proteins is elevated ten-fold, dearly showing the biochemical consequences of expression of the src tyrosine protein kinase. Members of the tyrosine kinase family of oncogenes all encode proteins that exhibit tyrosine protein kinase activity. Sequence analysis has revealed the functional relatedness of this family of oncoproteins, in that they all contain a conserved tyrosine kinase domain flanked by divergent domains presumably required to define the “targets” of the kinase activity and regulate enzyme activity.

Oncogene encoded proteins belonging to the tyro sine kinase family that span the plasma membrane are related to specific receptor molecules, such as the EGF receptor, the PDGF receptor, and the insulin receptor.

The functional relationship between these proteins and growth factor/mitogen receptors has been confirmed by the demonstration that the ammo acid sequence of the erb-B gene product is virtually identical to a portion of the EGF receptor and represents a truncated version of the EGF receptor. It is likely that the membrane-spanning tyrosine kinase oncoproteins induce cellular transformation by interrupting or altering in some way signal transduction across the plasma membrane. The exact mechanism by which the src protein and its close relatives induce transformation is less clear. However, their presence on the inner face of the plasma membrane suggests that these proteins may be “coupled” to other “receptor-like” molecules and may play a role in transmitting cellular signals in response to extracellular stimuli. Several cellular proteins that interact directly or indirectly with the src protein have been identified.

The proteins encoded by the oncogenes mos, raf, mil, and rel exhibit distant, but clearly identifiable, homology to the tyrosine kinase family of proteins. At least two members of this family, the raf and mos proteins, possess an associated serine/threonine kinase activity. The proteins in this family also are likely to exert their oncogenic effects by altering the pattern of serine/threonine phosphorylation in transformed cells.

THE RAS FAMILY

The third family of cytoplasmic oncoproteins is made up of three highly conserved proteins encoded by three distinct ras genes in humans (see Table 3). The human Ha raj-gene is the homologue of the oncogene of Harvey murine sarcoma virus, whereas the human K-ras gene is the homologue of the oncogene of the Kirsten murine sarcoma virus. N-ras, the third member of this gene family, was identified in human tumour cells using DNA transfection techniques (sec later). Each ras gene encodes a protein of 21 kilodaltons, p21, which is associated with the inner face of the plasma membrane. The ras-proteins are not protein kinases but instead bind guanine nucleotides, both GDP and GTP. Studies have shown that the ras proteins have the capacity to hydrolyze GTP to GDP, a property shared with several other known GTP binding proteins that function as coupling factors in hormone mediated signalling. Interestingly, the transforming ras proteins encoded by either the Ha ras or K ras genes (or by activated cellular ras genes, sec Fig 11.B) have an impaired ability to hydrolyze GTP compared with the ras protein encoded by a normal cellular ras gene. This is the result of a single nucleotide change in the normal cellular ras-gene. The structural and functional similarities between ras-proteins and coupling factors prompt conjecture that normal cellular p21^ras has a receptor-coupling factor function, and that the activated oncogenic version may transmit a continuous signal rather than a highly regulated one.

NUCLEAR ONCOGENES. Several oncoproteins appear to mediate cellular transformation by altering events taking place in the nucleus. The role of these gene products in the regulation of gene transcription has been suggested by several experimental results. Among the most provocative are experiments in which the treatment of quiescent cells with mitogens or growth factors results in the rapid induction of fos and myc gene expression. In addition, it is clear that enhanced expression of the myc gene in certain mouse and human tumours results in sustained growth of the tumour in the animal. These results suggest that alterations in the expression of gene products that normally control or regulate gene expression (nuclear proto-oncogenes) can provoke stable cellular transformation.

The continued characterization of nuclear oncoproteins, particularly fos and jun (the so-called fos-jun connection) has provided unique insights into how gene transcription is altered in transformed cells. The fos oncogene was first identified as the oncogenic component of a retrovirus that caused osteosarcomas in mice. The jun oncogene was initially found within the genome of the avian
retrovirus, ASV 17, a virus that causes fibrosarcomas in chickens. The first clue regarding the function of the jun oncogene came from the observation that the ammo acid sequence of v-jun was strikingly similar to that of a cellular transcription factor called AP 1. AP-1 stimulates the transcription of genes that carry an AP-1-binding site up stream from the cellular transcnptional promoter. Hence, it was concluded that v-jun triggers transformation by promoting the unregulated expression of certain cellular genes The role of the fos protein in the control of gene expression became apparent when it was recognized that the fos protein formed stable complexes with a cellular protein This protein is the cellular homologue of jun. Therefore, v-fos appears to induce transformation by binding to c-jun (a cellular transcription factor) and presumably altering gene expression. The recurring theme in all aspects of oncogene study is that cellular signalling pathways that result in altered gene expression are well paved with oncogene products.

Proto-Oncogenes and Oncogenes: Mechanisms of Activation

Acute transforming retroviruses induce transformation because they express a “captured” cellular oncogene sequence In addition, it is well documented that nonacute retroviruses can transcriptionally activate cellular proto-oncogenes by insertional mutagenesis, thereby inducing cellular transformation. Proto-oncogenes also can be activated by mechanisms other than retrovirus integration. Three such mechanisms have been described- somatic mutations, chromosomal translocations, and proto-oncogene amplification.

PROTO-ONCOGENE MUTATIONS. The first clues indicating that cells transformed by agents other than viruses contained activated oncogenes came from experiments using the techniques of DNA transfection. In these experiments, investigators prepared DNA from several human tumour cell lines and applied this DNA to normal mouse cell lines in culture (NIH 3T3 cells). It was observed that DNA from certain tumours induced the formation of foci or patches of transformed NIH 3T3 cells at a frequency much greater than that generated by DNA from normal cells (Fig.). If DNA was prepared from transformed recipient cells and used in a second experiment to artificially infect normal NIH 3T3 cells, an elevated frequency of transformed foci again was observed. Finally, if cells derived from such transformed foci were injected into mice, tumours were observed. The ability to detect and transfer genes capable of inducing transformation of NIH 3T3 cells led rapidly to the molecular cloning and characterization of the genes responsible for such transformation. DNA sequence analysis of the first gene that was cloned in this way showed that it was identical to the ras gene of the Harvey sarcoma virus (Ha ras; see earlier). In addition, the activated human ras gene differed from its normal cellular homologue at a single position, the codon for ammo acid 12, the precise position at which the Ha-ras gene was altered.

 

FIGURE. Schematic diagram of the steps required for the molecular cloning ot oncogenes from human tumour cell DNA. See text for details

 

These data, as well as other evidence, corroborate the conclusion that a single codon mutation in the sequence of the human Ha ras gene is sufficient to activate this proto-oncogene Many other human tumour cells have now been examined for activated oncogenes, using the DNA transfection method. Soon after the isolation of Ha-ras, such experiments identified two other oncogenes present m human tumour cells: the K-ras gene (structurally identical to the K-ras gene of Kirsten sarcoma virus) and the N-ras gene (structurally similar to Ha-ras and K-ras). Both the activated human K ras and N ras oncogenes encode proteins containing a single amino acid change compared with their normal cellular homologues. Although it is difficult to ascertain directly the role of the activated ras gene in the etiology of the human tumour in which it is found, the presence of a specific mutation suggests that such a change is likely to be an important step in the genesis of the tumour.

The search for additional human oncogenes using the NIH-3T3 DNA transfection assay has led to the identification of several other putative oncogenes.

CHROMOSOMAL TRANSLOCATIONS, NEOPLASIA, AND ONCOGENE ACTIVATION

The association between specific chromosomal translocations and certain humaeoplasms has been recognized for many years. Consistent chromosomal aberrations are found primarily in hematologic malignancies, as well as in tumours of embryonic origin. Specific translocations are a prominent feature of many types of leukemia and lymphoma Translocations are principally of two types (1) constitutional (ie, carried by all cells of the affected individual); and (2) somatic (ie, alterations that occur in a particular cell and are carried only by its neoplastic progeny). Constitutional translocations (and other chromosomal abnormalities) are thought to predispose an organism to the development of a malignancy, but this r squires additional mutations to fix the malignant transformation. An example of this is hereditary renal cell carcinoma, in which a constitutional translocation involving chromosomes 3 and 8 is associated with the development of renal cell carcinoma during the fourth decade of life.

Somatic translocations, on the other hand, arise in a single cell and contribute in some way to the malignant transformation of that initial cell and its progeny. Although it is difficult to assess the precise role of the translocation in the neoplastic process, the close association between particular  malignancies and specific translocations (Table) makes a strong, yet somewhat circumstantial, argument that these translocations are causally related to the development of the neoplasm.

Neoplasms With Consistent Chromosomal Defects

 

  * Few cases reported

 

The localization of proto-oncogene sequences on various chromosomes of humans and mice led to the recognition that somatic translocations associated with certaieoplasms involved chromosomal segments contaming proto-oncogene sequences. Such discoveries suggested the possibility that translocations of a proto-oncogene sequence may transcriptionally activate proto-oncogene expression or perhaps alter its gene structure.

One of the better understood examples of tumour-associated chromosomal translocation is that of Burkitt’s lymphoma (BL) Three characteristic somatic translocations are associated with BL, 90% of the tumours have a reciprocal translocation involving the long arms of chromosomes 8 and 14 (t8;tl4). The remaining tumours contain translocations involving chromosome 8 and either chromosome 2 (t2;t8) or chromosome 22 (t8;t22). Molecular cloning and DNA sequence analysis have demonstrated that in BL, the chromosomal translocations result in the juxtaposition of the c-myc proto-oncogene to a portion of the immunoglobulin heavy-chain gene (t8,tl4) or to the k (t2;t8) or A light-chain (t8;t22) genes (Fig.).

 

FIGURE. Chromosomal rearrangements involved in Burkitt’s lymphoma The human chromosomes 2, 14, and 22 are depicted with the positions of the Igκ, IgH, and Igλ genes Chromosome 8 also is shown with the position of the c-myc gene. The arrows denote the positions of the various breakpoints observed in reciprocal translocations involving chromosome 8 and chromosome 14, 2, or 22.

 

 

In the case of t8;tl4 translocations, the c-myc–gene resides adjacent to the break point and is transcribed in the direction opposite from that of the heavy-chain gene. In most cases, the break point occurs in the switch region of the heavy chain gene locus, although some variability is observed. DNA sequences from the variable region of the immunoglobulin heavy chain gene also are found on chromosome 8, indicating that the chromosomal exchange is reciprocal.

 

 

The translocation of the c-myc gene to chromosome 14 can result in an enhancement of transcription of the translocated c-myc gene. Because the absolute level of c-myc expression is different in individual cases of BL, it is not clear what mechanisms modulate myc expression. However, the consistent association of c-myc translocation and BL leaves little doubt that the transcriptional activation of the myc gene is an important prerequisite step in the development of BL.

Several other observations suggest that proto-oncogene expression may be activated by specific chromosomal translocations. The proto-oncogene c-mos is located on chromosome 8 near the break point observed in the translocation (t8;t21) associated with myeloblastic leukemia. In chronic myelogenous leukemia, the c-abl proto-oncogene, normally present on chromosome 9, is translocated to chromosome 22, resulting in an aberrant chromosome designated the Philadelphia (22q-) chromosome. Chronic myelogenous leukemia cells express an aberrant form of the c-abl protein, suggesting that the t9;t22 translocation gives rise to the expression of an altered oncogene product. Finally, in follicular lymphoma cells (a B-cell neoplasm), the characteristic translocation involving chromosomes 14 and 18 has been examined. In these cells, a portion of chromosome 18 is translocated to the immunoglobulin heavy chain gene in a manner similar to the translocations observed in BL. The translocated gene locus on chromosome 18 is unrelated to any known oncogene and may represent a novel oncogene important for the pathogenesis of B cell neoplasms.

In summary, it is clear that oncogene activation and somatic translocations go hand in hand with the process of neoplastic transformation. In addition, continued study of the gene sequences involved in unique translocations should provide a new dimension to our understanding of neoplastic development.

CHROMOSOMAL AMPLIFICATION. Solid tumours often contain chromosomes with homogeneous staining regions and acentric chromosomal fragments termed double minutes. This chromosomal material has been shown to be the result of gene amplification. Of special interest is the gene amplification observed in a particular subset of small cell lung carcinoma (SCLC), termed variant SCLC (SCLC V). Patients with SCLC-V have an inferior response to chemotherapy and radiotherapy and a much shorter survival than do other patients with SCLC. SCLC V cell lines often have double minutes and homogeneous staining regions, and an analysis of proto-oncogene levels in these cells has revealed a 20- to 40-fold amplification of c-myc and L- myc (a closely related myc gene) DNA sequences. The increased copy number of myc genes is accompanied by a commensurate increase in the level of myc RNA and protein expression. These observations suggest that myc-gene amplification plays a role in the development of this type of tumour.

An analogous amplification of a second gene structurally related to c-myc, termed the N-myc gene, has been observed in many human  neuroblastoma cell lines and in some SCLC-V tumour lines. The amplification of these genes indicates that they play an important role in the malignant progression of certain types of neuroblastomas and carcinomas.

AUTOCRINE GROWTH FACTORS. Evolving knowledge about the role of growth factors, mitogens, receptors, and oncogenes has focused intense interest on the possible role of these factors in the establishment of the neoplastic phenotype. The relatively autonomous nature of malignant cells has been known for many years. For example, tumour cells require fewer exogenous growth factors for optimal growth and multiplication than do their normal counterparts. To help explain this phenomenon, it has been suggested that transformed cells produce polypeptide growth factors, which, in turn, act on their own functional external receptors, thereby exerting the effect of the polypeptide growth factor on the same cell that produces it. Such a process has been designated autocrine stimulation.

Many types of tumour cells release polypeptide growth factors into the medium when grown in cell culture. These tumour cells usually possess receptors for the released factor. Several peptide growth factors have been demonstrated to function through the autocrine stimulation mechanism. These include transforming growth factor-a, peptides related to PDGF, bombesin, and transforming growth factor b. The activity of each of these four growth factors, and likely many others, is mediated by a different cell surface receptor. Activation of the receptor triggers a signalling mechanism that eventually leads to a mitogenic response. Support for the autocrine hypothesis derives in large part from experiments establishing the structural relationship between the oncogene of simian sarcoma virus v-sis and PDGF. The amino-terminal 109 amino acids of the b chain of PDGF are almost identical to the amino acid sequence of the v-sis gene product, p28^v-sis. Therefore, the oncogenicity of simian sarcoma virus is related directly to the ability of this virus to express a PDGF-like growth factor.

These observations lend further support to the concept that proteins encoded by oncogenes (or  inappropriately expressed or mutated proto-oncogenes) can function at several points in the cellular signalling cascade. Some oncoproteins confer growth-factor autonomy, some appear to activate postreceptor signalling pathways, and some lead to an alteration in synthesis and the release of a specific growth factor. Therefore, a cancer cell is likely to generally be a product of one or more genetic events that lead to profound changes in growth control.

Recessive Oncogenes: Antioncogenes

As long ago as the middle of the last century, it was observed that certain forms of cancer can cluster in families. In such cases, the genetic predisposition to cancer often behaves as an autosomal dominant trait. The childhood cancer retinoblastoma, a tumour of the eye, occurs in two forms: heritable, characterized by its autosomal inheritance within families, and sporadic, arising in children with no family history of disease or apparent risks. Epidemiologic studies led to the hypothesis that heritableretinoblastoma results from the inheritance of a predisposing mutation from the affected parent, and that, under normal circumstances (ie, in most cells), the mutation itself was not sufficient to induce the cancer (ie, it behaved  like a recessive mutation). Genetic studies at the molecular level have now shown that the original hypothesis was correct. Retinoblastoma tumour cells have a characteristic genetic abnormality in that chromosome 13 always contains a deletion of band 14, designated the Rb1 locus. On the other hand, normal cells from the same individual are heterozygous for this locus, containing one unaltered chromosome 13 and one copy of chromosome 13 with a deleted Rb1 locus (Fig.).

 

 

FIGURE. Diagram of the genetic events leading to retinoblastoma. Filled boxes denote deletions of the Rb 1 locus.

 

One hypothesis to explain the appearance of the tumour is that the wild-type Rb1 allele serves to suppress the tumour phenotype, and that the rare genetic events that give rise to the loss of that allele result in the formation of tumour cells. This would occur by chance much more frequently in an individual who was genetically heterozygous for this allele than in a homozygous person. Hence, heritable retinoblastoma is much more common than is the rare sporadic form, in which both alleles must be lost. The concept that certain genes, such as the Rb gene, can suppress tumour formation has led investigators to coin the term antioncogenes, or tumour suppressor genes, to designate genes that act in a positive way to promote the normal growth of cells. The observation that the introduction of a cloned Rb gene into retinoblastoma or osteosarcoma cell cultures suppresses the neoplastic phenotype strongly supports its role as an antioncogene.

The cellular gene known as p53 is another example of a tumor suppressor gene that is capable of counteracting transformation. It has been shown that p53 mutations are common in certain human cancers (eg, certain forms of colon cancer). Evidence suggests that both the p53 protein and the Rb gene product play important roles in cell-cycle regulation.

Several lines of evidence indicate that the protein products of antioncogenes and oncogenes may interact functionally to promote changes in cell growth leading to transformation. For example, in cells transformed by the DNA virus, SV40, the protein encoded by the transforming oncogene of this virus, large T antigen, is found to be tightly associated with the product of the normal Rb gene. Mutations in the SV40 oncogene that block the transformation of cells by SV40 also change the structure of the T-antigen protein such that it no longer binds to the normal Rb protein. Large T antigen also binds to the p53 protein, and this interaction is important for transformation as well. As described in Chapter 41, the oncogene products of adenoviruses as well as oncogenic papillomaviruses also bind to the same two cellular proteins. Thus, it is likely that these viruses transform cells at least partially by preventing (or inhibiting) the action of these tumour suppressor proteins.

 

ELISA for laboratory diagnosis of HIV-carrier.

 

 

Western blott

 

Prions. Slow infection

 

A proteinaceous infectious particle, or prion, is an infectious agent composed primarily of protein.^[2] The word prion, coined in 1982 by Dr. Stanley B. Prusiner, is a portmanteau derived from the words protein and infection.^[3] Prions are the cause of a number of diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as “mad cow disease”) in cattle and Creutzfeldt–Jakob disease (CJD) in humans. In general usage, prion refers to the theoretical unit of infection. All known prion diseases affect the structure of the brain or other neural tissue and all are currently untreatable and universally fatal.

Prions propagate by transmitting a mis-folded protein state: so as with viruses the protein cannot replicate by itself. Instead, when a prion enters a healthy organism the prion form of a protein induces pre-existing normal forms of the protein to convert into the rogue form. Since the new prions can then go on to convert more proteins themselves, this triggers a chain reaction that produces large amounts of the prion form. All known prions induce the formation of an amyloid fold, in which the protein polymerises into an aggregate consisting of tightly packed beta sheets. Amyloid aggregates are fibrils, growing at their ends, and replicating when breakage causes two growing ends to become four growing ends. The incubation period of prion diseases is determined by the exponential growth rate associated with prion replication, which is a balance between the linear growth and the breakage of aggregates.

This altered structure is extremely stable and accumulates in infected tissue, causing tissue damage and cell death. This structural stability means that prions are resistant to denaturation by chemical and physical agents, making disposal and containment of these particles difficult. Prions come in different strains, each with a slightly different structure, and most of the time, strains breed true. Prion replication is nevertheless subject to occasional epimutation and then natural selection just like other forms of replication. However, the number of possible distinct prion strains is likely far smaller than the number of possible DNA sequences, so evolution takes place within a limited space.

In scientific notation, PrP^C refers to the endogenous form of prion protein (PrP), which is found in a multitude of tissues, while PrP^Sc refers to the misfolded form of PrP, that is responsible for the formation of amyloid plaques and neurodegeneration. The precise structure of the prion is not known, though they can be formed by combining PrP^C, polyadenylic acid, and lipids. Proteins showing prion-type behavior are also found in some fungi, which has been useful in helping to understand mammalian prions. Fungal prions, however, do not appear to cause disease in their hosts and may even confer an evolutionary advantage through a form of protein-based inheritance.

 

Discovery

Radiation biologist Tikvah Alper and mathematician John Stanley Griffith developed the hypothesis during the 1960s that some transmissible spongiform encephalopathies are caused by an infectious agent consisting solely of proteins. Their theory was developed to explain the discovery that the mysterious infectious agent causing the diseases scrapie and Creutzfeldt–Jakob disease resisted ultraviolet radiation. Francis Crick recognized the potential importance of the Griffith protein-only hypothesis for scrapie propagation in the second edition of his “Central dogma of molecular biology“: while asserting that the flow of sequence information from protein to protein, or from protein to RNA and DNA was “precluded”. He noted that Griffith’s hypothesis was a potential contradiction (although it was not so promoted by Griffith). The revised hypothesis was later formulated, in part, to accommodate discovery of reverse transcription by Howard Temin and David Baltimore.

Stanley B. Prusiner of the University of California, San Francisco announced in 1982 that his team had purified the hypothetical infectious prion, and that the infectious agent consisted mainly of a specific protein – though they did not manage to isolate the protein until two years after Prusiner’s announcement. Prusiner coined the word “prion” as a name for the infectious agent. While the infectious agent was named a prion, the specific protein that the prion was composed of is also known as the Prion Protein (PrP), though this protein may occur both in infectious and non-infectious forms. Prusiner was awarded the Nobel Prize in Physiology or Medicine in 1997 for his research into prions.

Structure

Isoforms

The protein that prions are made of (PrP) is found throughout the body, even in healthy people and animals. However, PrP found in infectious material has a different structure and is resistant to proteases, the enzymes in the body that caormally break down proteins. The normal form of the protein is called PrP^C, while the infectious form is called PrP^Sc — the C refers to ‘cellular’ or ‘common’ PrP, while the Sc refers to ‘scrapie‘, a prion disease occurring in sheep. While PrP^C is structurally well-defined, PrP^Sc is certainly polydisperse and defined at a relatively poor level. PrP can be induced to fold into other more-or-less well-defined isoforms in vitro, and their relationship to the form(s) that are pathogenic in vivo is not yet clear.

Protein structure of the normal prion protein (PrP)

PrP^C

PrP^C is a normal protein found on the membranes of cells. It has 209 amino acids (in humans), one disulfide bond, a molecular weight of 35-36 kDa and a mainly alpha-helical structure. Several topological forms exist; one cell surface form anchored via glycolipid and two transmembrane forms. The normal protein is not sedimentable; meaning it cannot be separated by centrifuging techniques. Its function is a complex issue that continues to be investigated. PrP^C binds copper (II) ions with high affinity. The significance of this finding is not clear, but it presumably relates to PrP structure or function. PrP^C is readily digested by proteinase K and can be liberated from the cell surface in vitro by the enzyme phosphoinositide phospholipase C (PI-PLC), which cleaves the glycophosphatidylinositol (GPI) glycolipid anchor. PrP has been reported to play important roles in cell-cell adhesion and intracellular signaling in vivo, and may therefore be involved in cell-cell communication in the brain.

PrP^Sc

The infectious isoform of PrP, known as PrP^Sc, is able to convert normal PrP^C proteins into the infectious isoform by changing their conformation, or shape; this, in turn, alters the way the proteins interconnect. Although the exact 3D structure of PrP^Sc is not known, it has a higher proportion of β-sheet structure in place of the normal α-helix structure.Aggregations of these abnormal isoforms form highly structured amyloid fibers, which accumulate to form plaques. It is unclear if these aggregates are the cause of cell damage or are simply a side effect of the underlying disease process. The end of each fiber acts as a template onto which free protein molecules may attach, allowing the fiber to grow. Only PrP molecules with an identical amino acid sequence to the infectious PrP^Sc are incorporated into the growing fiber. Although this property is not strictly shared by other proteins considered prions. The sup35p was shown to be able to be incorporated into existing aggregations even when three of the five oligopeptide repeats normally present were deleted.

Function

It has been proposed that neurodegeneration caused by prions may be related to abnormal function of PrP. However, the physiological function of the prion protein remains a controversial matter. While data from in vitro experiments suggest many dissimilar roles, studies on PrP knockout mice have provided only limited information because these animals exhibit only minor abnormalities. In recent research done in mice, it was found that the cleavage of prions in peripheral nerves causes the activation of myelin repair in Schwann Cells. And that the lack of prions caused demyelination in those cells.

PrP and long-term memory

There is evidence that PrP may have a normal function in maintenance of long-term memory. Maglio and colleagues have shown that mice without the genes for normal cellular PrP protein have altered hippocampal long-term potentiation.

PrP and stem cell renewal

A 2006 article from the Whitehead Institute for Biomedical Research indicates that PrP expression on stem cells is necessary for an organism’s self-renewal of bone marrow. The study showed that all long-term hematopoietic stem cells expressed PrP on their cell membrane and that hematopoietic tissues with PrP-null stem cells exhibited increased sensitivity to cell depletion.

Prion disease

Main article: Transmissible spongiform encephalopathy

Prions cause neurodegenerative disease by aggregating extracellularly within the central nervous system to form plaques known as amyloid, which disrupt the normal tissue structure. This disruption is characterized by “holes” in the tissue with resultant spongy architecture due to the vacuole formation in the neurons.

Other histological changes include astrogliosis and the absence of an inflammatory reaction. While the incubation period for prion diseases is generally quite long, once symptoms appear the disease progresses rapidly, leading to brain damage and death. Neurodegenerative symptoms can include convulsions, dementia, ataxia (balance and coordination dysfunction), and behavioural or personality changes.

All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal. A vaccine has been developed in mice, however, that may provide insight into providing a vaccine in humans to resist prion infections.Additionally, in 2006 scientists announced that they had genetically engineered cattle lacking a necessary gene for prion production – thus theoretically making them immune to BSE, building on research indicating that mice lacking normally occurring prion protein are resistant to infection by scrapie prion protein.

Many different mammalian species can be affected by prion diseases, as the prion protein (PrP) is very similar in all mammals. Due to small differences in PrP between different species it is unusual for a prion disease to be transmitted from one species to another. The human prion disease variant Creutzfeldt-Jakob disease, however, is believed to be caused by a prion which typically infects cattle, causing Bovine spongiform encephalopathy and is transmitted through infected meat.

Transmission

It has been recognized that prion diseases can arise in three different ways: acquired, familial, or sporadic. It is often assumed that the diseased form directly interacts with the normal form to make it rearrange its structure. One idea, the “Protein X” hypothesis, is that an as-yet unidentified cellular protein (Protein X) enables the conversion of PrP^C to PrP^Sc by bringing a molecule of each of the two together into a complex.

Current research suggests that the primary method of infection in animals is through ingestion. It is thought that prions may be deposited in the environment through the remains of dead animals and via urine, saliva, and other body fluids. They may then linger in the soil by binding to clay and other minerals.

 Sterilization

Infectious particles possessing nucleic acid are dependent upon it to direct their continued replication. Prions, however, are infectious by their effect on normal versions of the protein. Sterilizing prions therefore involves the denaturation of the protein to a state where the molecule is no longer able to induce the abnormal folding of normal proteins. Prions are generally quite resistant to proteases, heat, radiation, and formalin treatments, although their infectivity can be reduced by such treatments. Effective prion decontamination relies upon protein hydrolysis or reduction or destruction of protein tertiary structure. Examples include bleach, caustic soda, and strong acidic detergents such as LpH. 134°C (274°F) for 18 minutes in a pressurized steam autoclave may not be enough to deactivate the agent of disease. Ozone sterilization is currently being studied as a potential method for prion denature and deactivation. Renaturation of a completely denatured prion to infectious status has not yet been achieved, however partially denatured prions can be renatured to an infective status under certain artificial conditions.

The World Health Organization recommends any of the following three procedures for the sterilization of all heat-resistant surgical instruments to ensure that they are not contaminated with prions:

1.     Immerse in a pan containing 1N NaOH and heat in a gravity-displacement autoclave at 121°C for 30 minutes; clean; rinse in water; and then perform routine sterilization processes.

2.     Immerse in 1N NaOH or sodium hypochlorite (20,000 parts per million available chlorine) for 1 hour; transfer instruments to water; heat in a gravity-displacement autoclave at 121°C for 1 hour; clean; and then perform routine sterilization processes.

3.     Immerse in 1N NaOH or sodium hypochlorite (20,000 parts per million available chlorine) for 1 hour; remove and rinse in water, then transfer to an open pan and heat in a gravity-displacement (121°C) or in a porous-load (134°C) autoclave for 1 hour; clean; and then perform routine sterilization processes.

Debate

Whether prions are the agent which causes disease or merely a symptom caused by a different agent is still debated by a minority of researchers. The following sections describe several alternative hypotheses: some pertain to the composition of the infectious agent (protein-only, protein with other components, virus, or other), while others pertain to its mechanism of reproduction.

Protein hypothesis

Prior to the discovery of prions, it was thought that all pathogens used nucleic acids to direct their replication. The “protein hypothesis” states that a protein structure can replicate without the use of nucleic acid. This was initially controversial as it contradicts the so-called “central dogma of molecular biology“, which describes nucleic acid as the central form of replicative information.

Evidence in favor of a protein hypothesis includes:

• No virus particles, bacteria, or fungi have been conclusively associated with prion diseases, although Saccharomyces cerevisiae has been known to be associated with infectious, yet non-lethal prions, such as Sup35p.

• No nucleic acid has been conclusively associated with infectivity; agent is resistant to ultraviolet radiation

• No immune response to infection

• PrP^Sc experimentally transmitted between one species and another results in PrP^Sc with the amino-acid sequence of the recipient species, suggesting that replication of the donor agent does not occur

• Familial prion disease occurs in families with a mutation in the PrP gene, and mice with PrP mutations develop prion disease despite controlled conditions where transmission is prevented

• Animals lacking PrP^C do not contract prion disease.

• Infectious prions can be formed de novo from purified non-infectious components, in the absence of gene-coding nucleic acids.

Genetic factors

A gene for the normal protein has been identified: the PRNP gene. In all inherited cases of prion disease, there is a mutation in the PRNP gene. Many different PRNP mutations have been identified and it is thought that the mutations somehow make PrP^C more likely to change spontaneously into the abnormal PrP^Sc form. Although this discovery puts a hole in the general prion hypothesis, that prions can only aggregate proteins of identical amino acid make up. These mutations can occur throughout the gene. Some mutations involve expansion of the octapeptide repeat region at the N-terminal of PrP. Other mutations that have been identified as a cause of inherited prion disease occur at positions 102, 117 & 198 (GSS), 178, 200, 210 & 232 (CJD) and 178 (Fatal Familial Insomnia, FFI). The cause of prion disease can be sporadic, genetic, and infectious, or a combination of these factors. For example, in order to have scrapie, both an infectious agent and a susceptible genotype need to be present.

Multi-component hypothesis

In 2007, biochemist Surachai Supattapone and his colleagues at Dartmouth College produced purified infectious prions de novo from defined components (PrP^C, co-purified lipids, and a synthetic polyanionic molecule). These researchers also showed that the polyanionic molecule required for prion formation was selectively incorporated into high-affinity complexes with PrP molecules, leading them to hypothesize that infectious prions may be composed of multiple host components, including PrP, lipid, and polyanionic molecules, rather than PrP^Sc alone.

In 2010, Jiyan Ma and colleagues at The Ohio State University produced infectious prions from a recipe of bacterially expressed recombinant PrP, POPG phospholipid, and RNA, further supporting the multi-component hypothesis. This finding is in contrast to studies that found minimal infectious prions produced from recombinant PrP alone.

Heavy metal poisoning hypothesis

Recent reports suggest that imbalance of brain metal homeostasis is a significant cause of PrP^Sc-associated neurotoxicity, though the underlying mechanisms are difficult to explain based on existing information. Proposed hypotheses include a functional role for PrP^C in metal metabolism, and loss of this function due to aggregation to the disease associated PrP^Sc form as the cause of brain metal imbalance. Other views suggest gain of toxic function by PrP^Sc due to sequestration of PrP^C-associated metals within the aggregates, resulting in the generation of redox-active PrP^Sc complexes. The physiological implications of some PrP^C-metal interactions are known, while others are still unclear. The pathological implications of PrP^C-metal interaction include metal-induced oxidative damage, and in some instances conversion of PrP^C to a PrP^Sc-like form.

Viral hypothesis

The protein-only hypothesis has been criticised by those who feel that the simplest explanation of the evidence to date is viral. For more than a decade, Yale University neuropathologist Laura Manuelidis has been proposing that prion diseases are caused instead by an unidentified “slow” virus. In January 2007, she and her colleagues published an article reporting to have found a virus in 10%, or less, of their scrapie-infected cells in culture.

The virion hypothesis states that TSEs are caused by a replicable informational molecule (which is likely to be a nucleic acid) bound to PrP. Many TSEs, including scrapie and BSE, show strains with specific and distinct biological properties, a feature which supporters of the virion hypothesis feel is not explained by prions.

Evidence in favor of a viral hypothesis includes:

• Strain variation: differences in prion infectivity, incubation, symptomology and progression among species resembles that seen between viruses, especially RNA viruses

• The long incubation and rapid onset of symptoms resembles lentiviruses, such as HIV-induced AIDS

• Viral-like particles that do not appear to be composed of PrP have been found in some of the cells of scrapie- or CJD-infected cell lines.

Recent studies propagating TSE infectivity in cell-free reactions and in purified component chemical reactions strongly suggest against TSE viral nature. More recently, using a similar defined recipe of multiple components (PrP, POPG lipid, RNA), Jiyan Ma and colleagues generated infectious prions from recombinant PrP expressed from E. coli, casting further doubt on the viral hypothesis.