ÐÓ°ÉÔ­´´

HIV, the virus that causes AIDS, is a new virus to infect humans. During
the relatively short time since its existence became known, HIV has caused
devastating illness in more than 200 000 people worldwide, including more
than 110 000 Americans. More than half of those infected with HIV have already
died.

AIDS is, so far, a fatal disease, characterised by the loss of essential
cells of the immune system known as T4 lymphocytes. In healthy people, T4
lymphocytes are instrumental in regulating the immune system, which protects
the body from invasion by infectious agents or from the emergence of certain
tumour cells. Because HIV infection destroys the body’s ability to defend
itself against other invading microbes, many AIDS patients die from so-called
‘opportunistic infections’ – those that take advantage of a weakened immune
system. In a unique and perhaps ironic way, HIV ravages the very cells that,
under normal circumstances, would orchestrate an immune response against
the virus.

A strong foundation of basic scientific information about immunology,
molecular biology and virology, gathered over the past few decades, has
given scientists unique opportunities to halt HIV’s devastating path. Indeed,
never before has science moved so rapidly to identify and describe a disease-causing
virus. Research is now focusing on the mechanisms by which HIV wreaks havoc
on the immune system. This will guide scientists looking for ways of treating
HIV infection, probably by interfering with the most crucial points in the
life cycle of the virus.

A variety of experiments throughout the world have looked at the mechanism
by which HIV destroys T4 cells. So far, this work suggests that the virus
may use several different ploys. HIV may kill the cells that it infects
directly when it reproduces. Alternatively, the virus may rely on indirect
methods, such as interfering with the normal metabolism of cells, inhibiting
the production of essential molecules such as growth factors, or by preventing
immune cells from recognising and fighting off infectious agents. Some innocent
‘bystander’ cells may be killed without even becoming infected.

One thing that is certain is that HIV gains entry into T4 lymphocytes
by attaching to a molecule called CD4 that is present on the surface of
the T4 cells. Proteins on the exterior envelope of the virus bind tightly
to the CD4 molecule; HIV then enters the cell by fusing to the cell’s membrane.
Once inside, HIV hijacks the cell’s genetic machinery, using it to reproduce
more viruses. Equipped with an enzyme called reverse transcriptase, HIV
transcribes its own genetic material, RNA, into DNA. (Because HIV converts
its genetic material ‘backwards’ from the normal route for cells and many
viruses, it is known as a retrovirus.) The genetic material from the virus,
now in the form of DNA, then inserts itself somewhere in the genome of the
host cell; once there, it is called a ‘provirus’.

Once integrated into the host cell’s genetic material, the provirus
may lie dormant for long periods before its DNA instructs the cell to make
new viruses. Eventually, some signal prompts the cell to transcribe the
viral DNA into RNA. This process, which scientists understand in only a
limited way, is a critical step in increasing the number of particles of
HIV in the body. Some of these newly made RNA molecules will serve as genetic
material for the new generation of HIV; others contain information for proteins
that form the virus’s ‘skeleton’. Once the cell has made the viral components,
new viruses assemble and bud from the cell’s surface (see Figure 1).

The information contained in the DNA derived from HIV is arranged in
genes: these are the keys to the virus’s ability to destroy the human immune
system. HIV’s genetic material contains much more information than that
of retroviruses known to infect other animals. Most other retroviruses possess
from three to five genes, but HIV has nine. They seem to have two basic
functions. They either contain information for the proteins that make up
the structural components of the virus; or they code for proteins that regulate
the virus’s ability to infect a cell, incorporate its genetic material into
the cell’s genome, or reproduce new viruses.

In addition to the regulatory and structural genes, HIV contains a string
of nucleic acids called the long terminal repeats (LTRs). As the name implies,
these genetic sequences appear at both ends of the viral genome and contain
many regions that help to regulate the production of viral proteins. ÐÓ°ÉÔ­´´s
liken these regions to ‘on-off’ switches; proteins from both the virus and
the cell can trigger these switches by binding to the LTR sequences.

With its intricate network of regulatory genes, HIV controls its own
ability to reproduce. One key to this could be the nef gene, which makes
a protein that some researchers believe suppresses the virus’s reproduction:
it may allow the virus to remain latent or to exist at low levels, hidden
from the immune system, for long periods. The nef gene may partially explain
why a long and variable time elapses between infection with HIV and the
appearance of disease. ÐÓ°ÉÔ­´´s have recently estimated that the average
incubation period for AIDS is nearly 10 years. While some people, particularly
children, can develop the disease within months after infection, nearly
half of those infected with HIV will be free of AIDS for 10 years or longer.
Researchers do not yet know how long the incubation period may be, or even
if all infected people will eventually become ill.

In most people, HIV will eventually begin to destroy T4 cells (see Figure
2). The number of T4 cells in the blood may slowly decline as HIV kills
them, both directly and indirectly. In laboratory experiments, HIV kills
cells directly as newly formed viruses bud from the cell, leaving tiny holes
in its outer membrane. Large numbers of budding viruses appear to weaken
the membrane, causing the cell to rupture and die.

ÐÓ°ÉÔ­´´s have also observed ways in which HIV can destroy large numbers
of T4 cells without actually infecting them. For example, in tissue cultures,
HIV-infected cells can join up with uninfected ‘bystander’ cells. During
the process of initial infection, the protein on the envelope of the virus
binds to the CD4 molecule on the surface of the T4 cell. Infected cells
also produce viral proteins on their surfaces, and these molecules can bind
to the CD4 molecules on uninfected cells. As a result, the membranes of
infected and uninfected cells can fuse. Their cytoplasm mixes and they form
giant cells with many nuclei, called syncytia (see Figure 3). In culture
dishes, these giant cells usually die within 48 hours. Some scientists believe
that, when people are infected with HIV, uninfected T4 lymphocytes adjacent
to infected cells may be eliminated, too, when syncytia form.

There are other ideas about how uninfected T4 cells may die. For example,
other laboratory experiments suggest that ‘protective’ responses by the
immune system, which aim to neutralise and so eliminate HIV, may be to blame.
Antibodies or immune cells that are programmed to eliminate cells infected
with HIV may mistakenly kill uninfected T4 cells to which viral proteins
have become bound.

Although loss of T4 lymphocytes is the main defect of the immune system
in AIDS, the system may start acting abnormally long before the T4 cells
begin to die off. Many investigators have found that T4 cells from apparently
healthy individuals infected with HIV are abnormal in several ways. First,
they cannot induce other immune cells called B lymphocytes to produce disease-fighting
antibodies. Secondly, the T4 cells fail to recognise foreign substances
or antigens and fail to proliferate, as they would do normally, in response
to them. Finally, the cells lose their ability to produce interleukin-2
(IL-2), a molecule that enhances the growth of T cells and helps to stimulate
their response to foreign antigens.

T cells from HIV-infected individuals also have abnormally low levels
of receptors for IL-2 on their surfaces. These receptors are the docking
sites that enable IL-2 to gain entry into the cell and stimulate the cell
to proliferate. ÐÓ°ÉÔ­´´s have reproduced the abnormal function of T4 cells
from HIV-infected individuals in the laboratory: when they exposed normal
T4 cells to non-infectious HIV or purified proteins from HIV, the T4 cells
were unable to respond to foreign antigens. Some scientists believe that
the reason why HIV causes abnormalities in the T4 cells may lie in the shape
of the virus’s envelope protein. Part of the protein resembles the IL-2
molecule, so it may fit into the docking site for IL-2 on the surface of
the T4 cell. This would prevent IL-2 from binding to the T4 cell and so
stimulating the cell’s response to antigens. HIV infection may also suppress
the function of normal human genes, particularly those that code for IL-2
and its receptors on the T4 cells.

Another possible mechanism by which HIV destroys immune functions includes
the interaction between T cells and other immune cells, called monocytes.
During a normal immune response, the monocyte’s job is to engulf and process
antigens, eventually shuttling fragments of the antigen back to the surface
of the cell. There, the antigen fragments are arranged within a group of
molecules called the class II major histocompatibility complex (MHC). The
main function of the MHC is to bring the ‘processed’ antigen in direct contact
with the receptor for antigens that is present on the T4 cell. The MHC achieves
this by attaching to the T4 cell’s CD4 receptor (although the actual receptor
for antigens on the T4 cell is distinct from the CD4 molecule). T4 cells
must come into contact with processed antigen before they can launch an
immune response against an invader. But, by binding to the T4 cell’s CD4
receptor, intact HIV or its envelope proteins may prevent the monocyte from
presenting antigens to the T4 cell. If a T4 cell is unable to ‘see’ processed
antigen, the immune response is cancelled (see opposite).

ÐÓ°ÉÔ­´´s have also found that cells infected with HIV in tissue culture
produce fewer CD4 molecules on their surfaces than do uninfected cells.
A decrease of this kind can also mean fewer chances for the monocyte to
interact with the T4 cell.

HIV infects not only T4 cells but other cells in the body as well. The
virus seems to be able to infect any cell that has CD4 molecules on its
surface. Besides T4 cells, the principal cells that HIV infects are monocytes,
which circulate in the blood, and macrophages, which are monocytes that
have migrated into body organs and taken on specific functions.

Researchers have discovered that the effect of HIV infection on T4 cells
is strikingly different to its effect on monocytes or macrophages. Unlike
T4 cells, monocytes and macrophages appear to be relatively resistant to
killing by HIV. In some cases, HIV even reproduces within the monocyte without
budding off from the outside cell membrane. Immature HIV particles acquire
their envelope protein coat by budding instead from membranes within the
cell. As a result, an infected monocyte may not release virus particles
into the surrounding tissue and may not have viral proteins projecting from
its surface.

Silent spread

By infecting monocytes, HIV finds safe haven from immune responses that
normally limit viral infections, while building up reservoirs of virus to
spread around the body. Monocytes travelling throughout the body can carry
HIV to various organs, particularly the lungs and the brain, and may damage
these tissues. People infected with HIV often experience symptoms resulting
from abnormalities in the central nervous system. While some of these are
undoubtedly caused by the many infections and tumours that afflict people
with impaired immune systems, scientists suspect that HIV, hitch-hiking
in monocytes, causes many of the neurological symptoms directly. Cells resident
in the brain, called microglial cells, may also be infected.

One of the most puzzling aspects of HIV infection is the fact that people
infected with this virus may not develop symptoms of immune deterioration
for many years. During that time, it is difficult to isolate HIV from an
infected person’s blood. But extremely sensitive tests show that viral DNA
is present in up to 1 per cent of circulating T4 cells. If we could understand
how HIV remains in this quiescent state and what triggers it to reproduce,
we might be able to design treatments to prevent the viral DNA in the host
cell from being activated. Even after viral DNA has become activated, therapies
may be able to drive the infection back into a latent state.

In early attempts to isolate an infectious agent from AIDS patients,
Robert Gallo, of the National Cancer Institute in Bethesda, Maryland, and
Luc Montagnier, of the Pasteur Institute in Paris, and their colleagues,
discovered that they could detect the viral enzyme reverse transcriptase
in lymphocytes if they grew the cells in media containing mitogens (substances
isolated from plants that promote cell growth) and certain other growth
factors. Later, other investigators observed that these growth factors and
mitogens increased the production of new viruses in HIV-infected lymphocytes
in cell culture. How do these substances trigger HIV to reproduce? It appears
that mitogens and certain growth factors prompt T cells to manufacture intracellular
activation factors that bind to the LTR of HIV and ‘turn on’ a reproduction
switch.

Mitogens are potent activators of HIV in the test tube, but it is unlikely
that they are responsible for stimulating the replication of the virus in
the body. Although mitogens are found naturally in several species of plants
(pokeweed, for example), scientists use them in experiments in a highly
purified and concentrated form. These concentrations would be far greater
than those to which human cells would be exposed, even if people ate these
plants.

Clearly, something is happening within the body of an HIV-infected person
to trigger the virus to replicate uncontrollably. To identify what factors
might help to activate HIV, the Laboratory of Immunoregulation at the National
Institute of Allergy and Infectious Diseases has examined whether exposing
infected T cells to antigens that people encounter in their daily lives
increased the production of HIV. When researchers exposed infected T cells
to soluble protein antigens such as tetanus toxin, they found that HIV replicated
at much higher levels than it did in T cells that had been stimulated with
antigens before infection with HIV.

Since many viruses can infect human cells, scientists have wondered
whether simultaneous infection with HIV and other viruses influences the
replication of HIV. To test this hypothesis, researchers linked the LTR
of HIV to a gene coding for an enzyme that is easy to measure in animal
cells. They then placed the composite gene into cells, together with the
regulatory genes from a range of viruses that frequently infect people infected
with HIV. The scientists found that the regulatory genes of herpes simplex
virus, cytomegalovirus, Epstein-Barr virus, hepatitis B virus, and human
herpesvirus type 6 all ‘turned on’ the LTR of HIV, directing production
of the marker enzyme. These results suggest that if certain other viruses
infect cells already infected with HIV, this may stimulate the cells to
make more HIV. Since herpes simplex virus, hepatitis B virus, human herpes
virus type 6 and HIV all infect the same cells in the body, the presence
of many different kinds of viruses may be partly responsible for activating
HIV in infected people.

Regulation and Activation

Researchers believe that, like mitogens, the regulatory genes of other
viruses interact with the LTR of HIV, either directly or by inducing activation
signals that the infected cell normally makes during its day-to-day operation.
This raises the issue of whether an HIV-infected cell produces viruses any
time it receives an activation signal telling it to carry out normal functions.

To answer this question, investigators in the Laboratory of Immunoregulation
established two cell lines, one from monocytes and the other from T cells.
Both cell lines were infected with HIV but produced very small amounts of
virus. The investigators then tested substances called cytokines for their
ability to spur on the cells to produce more HIV. (Cytokines, secreted by
T cells and monocytes during normal immune responses, alert immune cells
that an invader is present and recruit the cells to fight. IL-2 is a cytokine,
for example.) As predicted, certain cytokines did increase HIV production
in the cell lines. One of them, called tumour necrosis factor-alpha (TNF-alpha),
is made by monocytes present in the blood and in the fluid that bathes the
central nervous system. In someone infected with HIV, the TNF-alpha made
in response to ordinary viral and bacterial infections could activate HIV
to reproduce, gradually impairing the function of T4 cells and other normal
immune responses. Once the function of the T4 cells declines too far, the
body is susceptible to frequent and serious infections. In response to these
infections, cells produce TNF-alpha in even greater quantities which, in
turn, may cause further production of HIV, leading to the death of more
T4 cells and to neurological abnormalities. This self-sustaining cycle,
then, could ultimately lead to complete destruction of T4 cells, uncontrollable
infections and tumours, and death of the individual. We are not yet certain
if TNF-alpha plays an important role in stimulating HIV replication. There
may be many other forces acting on HIV that we have yet to identify.

Although scientists have a great deal more to learn about how HIV wreaks
havoc upon the human body, researchers worldwide have learnt an extraordinary
amount in a short time. Much of our success lies in the wealth of basic
research that took place before HIV or AIDS came onto the scene. Researchers
are now using this information to screen existing drugs and to design new
drugs to slow or halt the spread of the virus. In addition, researchers
are focusing their attention on ways to bolster the damaged immune system
and to treat more effectively the myriad opportunistic illnesses that afflict
people with impaired immune function. Lastly, we expect our efforts to understand
how latent infection with HIV develops into full-blown AIDS will shed light
on ways to prevent HIV from causing disease.

Dr Zeda Rosenberg is assistant to Dr Anthony Fauci, who is director
of the National Institute of Allergy and Infectious Diseases in Bethesda,
Maryland. Dr Fauci is also director of the Office of AIDS Research at the
National Institutes of Health in Bethesda.