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 in noncarriers. 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 in neonates 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 been named 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
Prevalence
of Delta Infection in Hepatitis B Virus (HBV) Carriers and
Persons
With HBsAg-Positive Acute and Chronic Hepatitis in
North Americae
|
Group
|
Number of Groups Studied
|
Delta Prevalence (%)
|
|
HBV carriers (blood donors)
|
15
|
1480
|
|
Acute hepatitis
|
6
|
15-72
|
|
Fulminant hepatitis
|
2
|
16-34
|
|
Chronic hepatitis
|
4
|
13-41
|
|
Cirrhosis
|
1
|
25
|
|
Primary hepatocellular
carcinoma
|
3
|
0-3
|
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 been named 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.
. 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.
Common serologic tests for HBV and their interpretation
|
Positive Tests
|
Interpretation
|
|
HBsAg
(surface antigen)
|
Current active hepatitis infection, acute or chronic
|
|
Anti-HBs (in
absence of HBsAg)
|
Protection against reinfection. Remains for years.
|
|
Anti-HBc (in
absence of anti-HBs)
|
Active HBV infection, acute or chronic. Can reveal active infection in
some instances when HBsAg is present at concentrations too low to be
detected.
|
|
HbeAg*
|
Active hepatitis infection, acute or chronic. Found in presence of HBsAg.
Indicates specimens that exhibit potential for enhanced infectivity.
|
|
Anti-HBe
|
When present in HBsAg carrier, blood is potentially less infectious.
|
*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 in nature 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.
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
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
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 initiation new 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.
Diagram of HIV
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 information needed 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 is carried out by another
viral enzyme called integrase.[42]
Reverse transcription of the HIV genome into double strand DNA
This integrated viral DNA may then lie dormant, in the latent stage of HIV
infection.[42] To actively produce the
virus, certain cellular transcription factors need to be present, the most important of which is NF-κB (NF kappa B), which is upregulated when T-cells become activated.[44] This means that those cells
most likely to be killed by HIV are those currently fighting infection.
During viral replication, the integrated DNA provirus is transcribed into mRNA, which is
then spliced into smaller pieces. These small pieces are exported from the nucleus into
the cytoplasm, where they are translated into the regulatory proteins Tat (which encourages new virus production) and Rev. As the newly produced Rev protein accumulates in the nucleus, it binds to
viral mRNAs and allows unspliced RNAs to leave the nucleus, where they are
otherwise retained until spliced.[45] At this stage, the structural
proteins Gag and Env are produced from the full-length mRNA. The full-length
RNA is actually the virus genome; it binds to the Gag protein and is packaged
into new virus particles.
HIV-1 and HIV-2 appear to package their RNA differently; HIV-1 will bind to
any appropriate RNA, whereas HIV-2 will preferentially bind to the mRNA that
was used to create the Gag protein itself. This may mean that HIV-1 is better
able to mutate (HIV-1 infection progresses to AIDS faster than HIV-2 infection
and is responsible for the majority of global infections).
HIV assembling on the surface of an infected macrophage.
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 HIV replication cycle
Entry to the cell
Genetic variability
HIV differs from many viruses in that it has very high genetic variability. This diversity is a result of its fast replication cycle, with the generation of about
1010 virions every day, coupled with a high mutation rate of approximately 3 x 10−5
per nucleotide base per cycle of replication and recombinogenic properties of reverse transcriptase.[48][49][50]
This complex scenario leads to the generation of many variants of HIV in a
single infected patient in the course of one day.[48] This variability is
compounded when a single cell is simultaneously infected by two or more
different strains of HIV. When simultaneous infection occurs, the genome of
progeny virions may be composed of RNA strands from two different strains. This
hybrid virion then infects a new cell where it undergoes replication. As this
happens, the reverse transcriptase, by jumping back and forth between the two
different RNA templates, will generate a newly synthesized retroviral DNA sequence that is a recombinant between
the two parental genomes.[48] This recombination is most
obvious when it occurs between subtypes.[48]
The closely related simian immunodeficiency
virus (SIV) has evolved into many strains, classified by the natural host
species. SIV strains of the African green monkey (SIVagm) and sooty mangabey (SIVsmm)
are thought to have a long evolutionary history with their hosts. These hosts
have adapted to the presence of the virus,[51] which is present at high
levels in the host's blood but evokes only a mild immune response,[52] does not cause the development of simian AIDS,[53] and does not undergo the extensive mutation and recombination typical of
HIV infection in humans.[54]
In contrast, when these strains infect species that have not adapted to SIV
("heterologous" hosts such as rhesus or cynomologus macaques), the
animals develop AIDS and the virus generates genetic diversity similar to what is seen in
human HIV infection.[55] Chimpanzee SIV (SIVcpz), the closest genetic relative of HIV-1, is
associated with increased mortality and AIDS-like symptoms in its natural host.[56] SIVcpz appears to have been
transmitted relatively recently to chimpanzee and human populations, so their
hosts have not yet adapted to the virus.[51] This virus has also lost a
function of the Nef gene that
is present in most SIVs; without this function, T cell depletion is more
likely, leading to immunodeficiency.[56]
Three groups of HIV-1 have been identified on the basis of differences in
the envelope (env) region: M, N, and O.[57] Group M is the most prevalent
and is subdivided into eight subtypes (or clades), based on the whole genome, which are geographically distinct.[58] The most
prevalent are subtypes B (found mainly in North America and Europe), A and D
(found mainly in Africa), and C (found mainly in Africa and Asia); these
subtypes form branches in the phylogenetic tree representing the lineage of the
M group of HIV-1. Coinfection with distinct subtypes gives rise to circulating
recombinant forms (CRFs). In 2000, the last year in which an analysis of global
subtype prevalence was made, 47.2% of infections worldwide were of subtype C,
26.7% were of subtype A/CRF02_AG, 12.3% were of subtype B, 5.3% were of subtype
D, 3.2% were of CRF_AE, and the remaining 5.3% were composed of other subtypes
and CRFs.[59] Most HIV-1 research is
focused on subtype B; few laboratories focus on the other subtypes.[60] The existence of a fourth
group, "P", has been hypothesised based on a virus isolated in 2009.[61] The strain is apparently
derived from gorilla SIV
(SIVgor), first isolated from western lowland gorillas in 2006.[61]
The genetic sequence of HIV-2 is only partially homologous to HIV-1 and
more closely resembles that of SIVsmm.
Reverse transcription of the HIV genome into double strand DNA
This integrated viral DNA may then lie dormant, in the latent stage of HIV
infection.[42] To actively produce the virus, certain cellular transcription factors need to be
present, the most important of which is NF-κB (NF kappa B), which is
upregulated when T-cells become activated.[44] This means that those cells most likely to be killed by HIV are those
currently fighting infection.
During viral replication, the integrated DNA provirus is transcribed into mRNA, which is then spliced into smaller pieces. These
small pieces are exported from the nucleus into the cytoplasm, where they are translated into the
regulatory proteins Tat (which encourages new virus production) and Rev. As the newly produced Rev protein accumulates in the nucleus, it binds to
viral mRNAs and allows unspliced RNAs to leave the nucleus, where they are otherwise
retained until spliced.[45] At this stage, the structural proteins Gag and Env are produced from the
full-length mRNA. The full-length RNA is actually the virus genome; it binds to
the Gag protein and is packaged into new virus particles.
HIV-1 and HIV-2 appear to package their RNA differently; HIV-1 will bind to
any appropriate RNA, whereas HIV-2 will preferentially bind to the mRNA that
was used to create the Gag protein itself. This may mean that HIV-1 is better
able to mutate (HIV-1 infection progresses to AIDS faster than HIV-2 infection
and is responsible for the majority of global infections).HIV assembling on the
surface of an infected macrophage.
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 2x109 HIV particles are produced each day
- An average of 2.6x109 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:
- 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).
- 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.
- 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.
Immune activation
and AIDS pathogenesis
http://www.google.com.ua/url?sa=t&rct=j&q=&esrc=s&source=web&cd=10&sqi=2&ved=0CJABEBYwCQ&url=http%3A%2F%2Fjournals.lww.com%2Faidsonline%2FFulltext%2F2008%2F02190%2FImmune_activation_and_AIDS_pathogenesis.1.aspx&ei=mZlAUfWpGM3Aswam2IDYDQ&usg=AFQjCNFwpg9ZA8-EXIbM_9vLUauUXAHu8Q&sig2=qjqF4nmJr0fRYcrlQlBBXA&bvm=bv.43287494,d.Yms&cad=rja
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 SIVmac239-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.
Back to Top | Article Outline
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.
Back to Top | Article Outline
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 in nature. 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 (Treg) 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 Treg in HIV
and SIV infection has been the subject of intense study over the past few years
[63-76]. Conceivably, Treg 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 Treg,
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
Image Tools
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 activation not 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].
Back to Top | Article Outline
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.
Back to Top | Article Outline
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 in natural 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].
Back to Top | Article Outline
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 SIVmac239-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.
Back to Top | Article Outline
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.
Back to Top | Article Outline
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 in nature. 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 (Treg) 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 Treg in HIV
and SIV infection has been the subject of intense study over the past few years
[63-76]. Conceivably, Treg 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 Treg,
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
Image Tools
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 activation not 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].
Back to Top | Article Outline
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.
Back to Top | Article Outline
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 in natural 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].
Back to Top | Article Outline
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
Pathogenesis of AIDS
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=rja
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 in nature,
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 in nature) 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 in normal 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.
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.
Figure. Structure 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 initiation new 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.
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.
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.
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.
Principal
Opportunistic Pathogens in AIDS Patients
|
Organism
|
Treatment
|
|
BACTERIA
|
|
|
Legionella pneumophila
Listena monocytogenes
Nocardia asteroides
Mycobacterium tuberculosis
|
Erythromycin
Ampicillin, penicillin G,
gentamicin Sulfonamide, trimethoprim-sulfamethoxazole Isoniazid + rifampin
+pyrazinamide + ethambutol
|
|
Mycobacterium avium-
intracellulare
|
Clarithromycin + ethambutol +
rifampin
|
|
Salmonella spp.
|
Ampicillin, chloramphenicot
|
|
Fungi
|
|
|
Candida spp
|
Amphotericin B, fluconazole
|
|
Coccidioides immilis
|
Amphotericin B, itraconazole
|
|
Cryptococcus neoformans
|
Amphotericiri B + flucytocine;
fluconazole
|
|
Histoplasma capsulatum
|
Amphotericin B, itracoriazole
|
|
Pneuinocystis carimi
|
Pentamidine,
trimethoprim-sulfamethoxazoie
|
|
protozoa
|
|
|
Toxoplasma gondii
|
Triple sulfonamides +
pyrimethamine
|
|
viruses
|
|
|
Herpes simplex
|
Acyclovir, foscamet
|
|
Cytomegalovirus
|
Ganciciovir, foscamet
|
|
Varicella-zoster
|
Acyclovir, foscamet
|
|
Measles
|
Supportive
|
|
JC virus
|
Supportive
|
|
Adenovirus
|
Supportive
|
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
hostgenes (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.
Viral
Oncogenes and Cellular Homologues of Known Function
|
Oncogene
|
Activity
|
Cellular
Homologue
|
|
Src
|
Tyrosine protein kinase
|
Membrane
associated tyrosine kinase
|
|
erb B
|
Tyrosine
protein kinase
|
Epidermal
growth factor receptor
|
|
fms
|
Tyrosine
protein kinase
|
Colony
stimulating factor
I receptor
|
|
sis
|
Growth
stimulation
|
Platelet
derived growth factor-B chain
|
|
raf
|
Serine/threonine
kinase
|
Cytoplasmic
receptor linked kinase
|
|
ras
|
GTP/GDP
binding
|
Receptor
coupling factors
|
|
Jun
|
DNA binding
|
Transcription
factor (AP-1 related)
|
|
erb A
|
DNA binding
|
Thyroxin receptor
|
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
|
Oncogene
|
Virus
|
Animal
of Origin
|
Virus Disease
|
Protein Product
|
Activity
|
|
src
|
RSV
|
Chicken
|
Sarcoma
|
pp60src
|
Tyrosine
kinase
|
|
fps/fes
|
Fujinami
ASV/ST FeSV
|
Chicken
|
Sarcoma
|
pp140gag-fps
|
Tyrosine
kinase
|
|
|
|
Cat
|
Sarcoma
|
p85gag-fes
|
Tyrosine
kinase
|
|
yes
|
Y73 ASV
|
Chicken
|
Sarcoma
|
p90gag-yes
|
Tyrosine
kinase
|
|
ros
|
UR-2 ASV
|
Chicken
|
Sarcoma
|
p68gag-ros
|
Tyrosine
kinase
|
|
fgr
|
GR FeSV
|
Cat
|
Sarcoma
|
p70gag-fgr
|
Tyrosine
kinase
|
|
abl
|
Abelson MLV
|
Mouse
|
Pre-B-cell
leukemia
|
p160gag-abl
|
Tyrosine
kinase
|
|
fms
|
SM KSV
|
Cat
|
Sarcoma
|
p180gag-fms
|
Tvrosine
kinase
|
|
erb B
|
AFV H,
AVE-ES4
|
Chicken
|
Ery
throblastosis
|
p68erbB
|
Tvrosine
kinase
|
|
erb A
|
At-V FS4
|
Chicken
|
Ebrythroblastosis
|
p75gag-erbA
|
Nuclear, DNA
binding
|
|
myc
|
MC29
|
Chickcn
|
Myeloeytomatosis
|
p110gag-myc
|
Nuclear, DNA
binding
|
|
myb
|
AMV
|
Chicken
|
Myeloblastosis
|
p45myb
|
Nuclear, DNA
binding
|
|
fos
|
FBR MSV
|
Mouse
|
Osteosarcoma
|
p75gag-fos
|
Nuclear, DNA
binding
|
|
Jun
|
ASV 17
|
Chickcn
|
Ostcosarcoma
|
p65gag-jun
|
Nuclear, DNA
binding
|
|
H-ras
|
Harvey MSV
|
Rat
|
Sarcoma and
erythroleukemia
|
p21Hras
|
GTP/GDP
binding protein
|
|
K-ras
|
Kirsten MSV
|
Rat
|
Sarcoma and erythroleukemia
|
p21Kras"
|
GTP/GDP
binding protein
|
|
N-ras
|
Neuroblastoma*
|
Human
|
**
|
p21Nras
|
GTP/GDP
binding protein
|
|
raf/mil
|
3611 MSV
|
Mouse
|
Sarcoma
|
p75gag-raf
|
Serine kinase
|
|
|
MH 2
|
Chicken
|
Sarcoma
|
p100gag-mil
|
Serine kinase
|
|
mos
|
Moloney MSV
|
Mouse
|
Sarcoma
|
p37mos
|
Serine kinase
|
|
sis
|
SSV
|
Monkey
|
Sarcoma
|
p28sis
|
PDGF-B chain
|
|
|
P1I-FeSV
|
Cat
|
Sarcoma
|
p76gag-sis
|
PDGF-B chain
|
|
rel
|
REV*
|
Chicken
|
Reticuloendothehosis
|
p59v-rel
|
***
|
|
ets
|
E26
|
Chicken
|
Erythroblastosis
|
p135gag-myb-etc
|
***
|
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 pp60src,
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 in normal 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 p21ras 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
human neoplasms 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
|
Disease
|
Chromosomal Defect
|
|
UNIQUE CHROMOWMAI DEFECTS
|
|
Acute lymphocytic leukemia,
L2
|
T (4;11)(q21;q23)
|
|
Acute myelogenous leukemia,
M2
|
t(8;21)(q22.1;q22.3)
|
|
Acute promyelocytic
leukemia, M3
|
t(15;17)(q22;q11.2)
|
|
Acute myelomonocytic
leukemia, M4
|
inv(16)(p13.2;q22)
|
|
CONSISTENTLY SHARFD CHROMOSOMAI DEFECTS
|
|
Acute nonlymphocytic
leukemias, sub-types Ml, M2, M4, M5, M6
|
del(5)(q22;q23)
mde 17(q33;q36)
+8
|
|
Burkitt's lymphoma
|
t(8;14)(q24.1;q32.3)
|
|
Acute lymphocytic leukemia, L3
|
|
|
Small
noncleaved non Burkitt's lymphoma
|
|
|
Immunoblastic
lymphoma*
|
|
|
Acute
monocytic leukemia
|
t(9;11)(p22;q23)
|
|
Acute
myelomonocytic leukemia
|
|
|
Chronic
myelogenous leukemia
|
t(9;22)(q34.1,q11.2)
|
|
Acute
myelogenous leukemia, M1
|
|
|
Acute
lymphocytic leukemia, L1, L2
|
|
|
Chronic
lymphocytic leukemia
|
t(11;14)(ql3;32)*
|
|
Small cell
lymphocytic lymphoma (transformed to diffuse large cell lymphoma)
|
|
|
Chronic
lymphocytic lymphoma
|
+12
|
|
Small cell
lymphocytic lymphoma
|
|
|
Follicular
small cleaved cell lymphoma
|
t(14;18)(q32.3,q21.3)
|
|
Follicular
mixed cell lymphoma
|
|
|
Follicular
large cell lymphoma
|
|
* 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 certain neoplasms
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, p28v-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 tumour 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
References:
1. Ronald M.
Atlas. Microbiology in our World, 1995.
2. Handbook on Microbiology. Laboratory diagnosis of
Infectious Disease/ Ed by Yu.S. Krivoshein, 1989. – P. 210-212, 215-217.
3. Medical Microbiology and Immunology: Examination and
Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P.257- 266, 286-298.
4.
Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/
Lange Medical Publication, Los Altos, California, 2002. – P.403-417, 501-529.
