INFECTIOUS DISEASE

TURKISH

 

VIROLOGY - CHAPTER   SEVEN    

PART SEVEN

HUMAN IMMUNODEFICIENCY VIRUS AND AIDS  

Life Cycle of HIV

Dr Richard Hunt
Professor
Department of Pathology, Microbiology and Immunology
University of South Carolina School of Medicine

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Figure 15
HIV budding from an infected cell CDC

Figure 15a
Stages of HIV budding.
A. The internal proteins assemble at the cell membrane and are seen as an electron-dense coat on the inner surface.
B. The virus buds with the internal proteins in a horseshoe or doughnut shape.
C. The internal proteins are cleaved and condense to form a cone-shaped inner mass (the nucleocapsid). The arrow shows a virus that is still attached to the host cell. By this stage condensation has occurred.
D. This image shows the cones cut in several planes

Cells that are infected by HIV

HIV destroys CD4+ T4 cells specifically, causing the profound immuno-suppression that is the hallmark of AIDS. Some other cells harbor and replicate the virus without lysis or, in the case of dendritic cells, they may concentrate virus at the cell surface with little or no replication of the virus. The major HIV-infected cell types are shown in figure 15c.

CD4+ T4 helper cells

HIV leads to disease as a result of the depletion of CD4+ T4 helper cells and the consequent inability to fight opportunistic infections. T4 cells are, not surprisingly, the major cell type that is infected by the virus. Infected CD4+ T4 helper cells become targets for HIV-specific CD8+ killer cells but  also die from a variety of other causes (see section 10). During the early acute infection stage, mostly mucosal CD4+ T4 cells are lost, while during chronic infection that may last many years, CD4+ T4 cells generally proliferate and die as a result of immune activation and other factors.

Infected cells that are detectable in the patient in the chronic stage of infection are usually T4 memory cells whereas naive T cells exhibit infection at a much lower frequency. The HIV-infected patient has a higher frequency that normal of  proliferating T4 cells as a result of general immune activation and these cells are targets for HIV (which only infects activated CD4+ T cells). The general immune activation may, in part, result from the translocation of bacteria across the damaged gut mucosa. Thus, HIV induces a constant supply of its target cells leading to further rounds of replication and immune destruction. The fact that HIV targets HIV-activated T4 cells leads to the reduction of T4 cells that are specific to HIV, thereby depleting the arm of the immune system that controls replication of the virus.

As noted elsewhere, after activation by a specific antigen, T4 cells either die or become non-proliferating memory cells which are rapidly mobilized if the antigen is subsequently reencountered. This latent reservoir of infected T4 cells can survive for many years, even in the presence of the current anti-HIV drugs (HAART - highly active anti-retroviral therapy) that appear to suppress HIV replication completely. This is because when an infected T4 cell reverts to the resting, memory state, it no longer replicates virus (that is makes the viral proteins and genomic RNA) but the cell still harbors a DNA copy of HIV (the provirus) integrated into its chromosomes. On reactivation of the cells by antigen, viral replication resumes.

Natural Killer cells

These are also CD4+ T cells and interact with dendritic cells. In addition to CD4 antigen, they express the co-receptor CCR5 and are thus infected by those HIV strains that require CCR5 for entry into the cell.

CD8+ Killer T cells

These cells express low levels of CD4 antigen when they are activated and appear to be infected in small numbers by HIV in the later stages of disease. Naive CD8 cells do not express CD4 antigen and do not appear to be infected (although they do express the co-receptors).

Macrophages

Monocytes/macrophages express CD4 antigen (although in much lower amounts that T4 cells) and are infected by HIV. They may provide an important reservoir for the virus within the host and may be especially important in HAART-treated patients. Macrophages also bind HIV gp120 via syndecan, a proteoglycan containing heparan sulfate and via CD91 antigen which interacts with heat shock proteins that the virus acquired from the cell in which it was replicated. Macrophage-adsorbed virus can be passed to other cells including T4 cells.

Non-proliferating mature macrophages can support HIV production for a long time without being killed. There is no latency in these cells, the virus just buds. Cytokine production by the infected macrophages is also aberrant leading to a variety of secondary effects. The slim disease that is characteristic of HIV infections in Africa may result from macrophage cytokine disruption. This wasting is very reminiscent of Visna in sheep and Visna infections involve the macrophages. Not only are macrophages and macrophage-like cells infected via CD4 antigen; when virus particles are coated (opsonised) by anti-HIV antibodies they can be taken up by macrophages via Fc or complement receptors.

Cells of the nervous system

HIV infects oligodendrocytes, astrocytes, neurones, glial cells and brain macrophages. Macrophage-tropic forms are found in the cerebro-spinal fluid. HIV causes disease of the central nervous system  which may result from the small protein, Tat, that is encoded by the virus and which acts as a general transactivator of transcription. This protein binds to neural cells via CD91 antigen and is internalized. As a result, cell metabolism is affected (such as nitric oxide signaling). HIV is also thought to compromise blood-retinal barrier integrity.

HIV in the brain and in the cerebro-spinal fluid may be particularly resistant to chemotherapy because of the failure of anti-retroviral drugs to penetrate the blood-brain barrier.

Dendritic cells

Follicular dendritic cells (FDCs) are important in the biology of HIV. These are antigen-presenting cells that process antigen and present peptides to T cells. They are not readily infected by HIV, though they can be productively infected as a result of having low levels of HIV receptors (CD4 antigen and the co-receptors CCR5 and CXCR4 - see below). Importantly, these cells trap HIV on their surfaces since they possess a surface lectin (called dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin or DC-SIGN) that binds to the carbohydrate components of HIV gp120. Binding by DC-SIGN does not allow fusion of the membrane of the virus with the FDC (which requires CD4 antigen) and so infection does not occur by this route; however, this protein also participates in the association of FDCs with lymphocytes and clusters at the sites of FDC-lymphocyte interactions. Thus, the bound virus is concentrated just at the site of interaction of the FDC with the CD4+ cell (figure 15 b). Moreover, receptors and co-receptors for HIV on the T4 cell also seem to cluster here.

When HIV enters the body via the mucosal route (epithelia of the vagina, penis or rectum), it is bound by FDCs that migrate to the lymph nodes; here the FDCs present HIV to T4 cells, which become infected.

Figure 15c
Cells that are infected by HIV

Figure 15d
Entry of HIV via the mucosal route and transit via dendritic cells to the lymph nodes

CD4 antigen is the HIV receptor  

The apparent specificity of CD4⁺ cell infection observed early in the history of HIV and AIDS, together with the observation that T4 cells are depleted in disease (indeed, the course of disease in the patient is followed by CD4⁺ T cell levels), suggested that CD4 antigen might be the receptor for the virus. This was demonstrated by transfecting the CD4 antigen gene into CD4^- human cells (such as cervical carcinoma HeLa cells) and showing that they acquired the property of being able to be infected by HIV (figure 16). CD4 antigen is the major receptor for both HIV-1 and HIV-2 in T4 cells and most other cells that can be infected by HIV.

A co-receptor for infection by HIV

The experiment in which CD4 antigen is transfected into cells that then acquire the ability to be infected by HIV only works when the transfected cells are human. If we do the same experiment with mouse 3T3 cells, the virus can bind to the cell surface, via CD4 antigen, but no infection ensues. Thus, something more than CD4 antigen is necessary.

It was also discovered that some strains of HIV (those adapted for life in transformed T cells) could infect and replicate in activated human T cells but not in monocytes or macrophages. Conversely, those adapted for life in macrophages could not infect and replicate in transformed T cells. Yet both macrophages and T4 cells possess CD4 antigen. The differences in tropism of the viral strains mapped to the V3 region of Gp120 suggesting that molecules other than CD4 antigen have an important role in infection and this role is CD4⁺ cell type-specific.

Chemokine receptors seem to be the key to the gateway of the cell -- A family of proteins on the surface of immune cells

Chemokines are small secreted proteins that are chemotactic for cells in the immune system such as leukocytes which move up the gradient of chemokine secreted by another cell; thus, they control the temporal and spatial positioning of leukocytes during an immune response. Chemokines are divided into two groups according to a conserved dicysteine motif that they contain. These are the C-C group and the C-X-C group. They bind to the cell surface via receptor molecules that are integral membrane proteins that span the plasma membrane seven times (seven transmembrane receptors). The receptors are named for the type of cytokine that they bind (CCR- or CXCR-).

• CCR5. Several laboratories identified an essential co-receptor for those HIV strains involved in critical early stages of infection (these are the macrophage-tropic strains). All of these studies found CCR5 as the partner of CD4 in allowing entry into macrophages.    

Figure 17B
Attachment of HIV to a CD4+ cell. The outer domain of gp120 binds to the CD4 antigen. This leads to a conformational change in gp120 and a co-receptor binding site is exposed. This region of gp120 binds to the chemokine receptor. Binding to the chemokine receptor allows another conformational change to occur so that regions of the gp41 HIV protein interact to form a fusion domain that allows the viral and cell membrane to fuse. As a result the viral core enters the cytoplasm.

The discovery of this molecule as a co-receptor came from previous studies that showed that three chemokines secreted by CD8⁺ T lymphocytes (called RANTES, MIP-1a and MIP-1b), which are involved in inflammatory responses, are powerful suppressors of HIV infection, and especially macrophage-tropic HIV. Of course, this suggested that the suppression of infection might be because the chemokines bound to the cell surface and blocked a receptor that HIV needed for entry. Suspicion fell on CCR5 because it was known to be the receptor that binds all three of the above chemokines (figure 17a).

• CXCR4 (also known as fusin). This is also a co-receptor for HIV in otherwise non-infectable CD4+ cells. CXCR4 is a G protein-coupled receptor whose ligand is a B cell stimulatory factor--it is called fusin because it promotes the infection/fusion of CD4+ cells.  The amount of CXCR4 on the cell surface may explain differences in tropism and CXCR4 seems to be more active in T cells than in macrophages. The CXCR4 gene most closely resembles the gene for IL-8 receptor (also a chemokine receptor)
   

• CCR2. This is another co-receptor.

Complexes of pieces of CD4 and Gp120 also bind to CCR5 on CD4^- cells. This explains why soluble CD4 actually enhances HIV infectivity rather than blocking it. It seems that Gp120 binds CD4 and undergoes a conformational change that increases its affinity for the chemokine receptor. The binding of the chemokine receptor causes a conformational change in the gp41 fusion protein of HIV that allows fusion of the viral membrane with the membrane of the cell to be infected. In fact, it is really the chemokine receptor that is the primary receptor for HIV and the role of CD4 is to concentrate virus at the cell surface and facilitate interaction with the chemokine receptor (figure 17b). In contrast to examples of CD4-independent HIV entry into cells, there are (so far) no examples of entry independent of chemokine receptors.

These co-receptors may explain the phenotypic switch during infection (see below). Changes in the amino acid sequence of Gp120 occur in the progression of the disease. It is likely that HIV uses CCR5 in the early stages of disease and then switches to CXCR4, perhaps avoiding the suppressive activity of chemokines. This also explains the transition from non-syncytium-inducing to syncytium-inducing phenotype. CXCR4 and CCR5 are members of a large family of receptors and the spread of HIV through subtypes of T cells may reflect subtle changes on the variable loops of Gp120 allowing the infection of new CD4⁺ cells with different receptors. This may also be one reason why so few CD4⁺ cells appear to be infected at any one time.

Some CD4-negative cells can be infected by HIV 

It was originally thought that only cells that have CD4 antigen can be infected by HIV. Although CD4 protein had not been demonstrated on some infectable cells, it was thought to be present in low amounts and CD4 antigen mRNA could be detected in most infectable cells. Specificity to CD4 positive cells reflects the specific binding of Gp120 to CD4. It is now known, however, that some non-CD4 cells, including some in brain and intestine, can be infected in a via a galactocerebroside receptor. Other cells can be infected in a different way; for example, in macrophages (see below) an Fc or complement receptor may be used. In these cases, the HIV must be bound by anti-HIV antibodies that interact with receptors on the cell surface. Thus anything that can up-regulate Fc receptors on macrophages will augment infection.

Entry into cell: pH-independent fusion with plasma membrane

No pH-dependent conformational change in a viral membrane protein is necessary for fusion between the viral membrane and the membrane of the cell to be infected. Thus, no entry into endosomes or lysosomes is required.

As with herpes virus, this sort of fusion of a virus with the plasma membrane is associated with fusions of infected cells to form syncytia. Syncytium formation is also a characteristic of HIV infection (figure 18). This has profound significance for spread of infection between cells without any free virus. This means that virus may spread from cell to cell so that immune system circulatory antibodies cannot have any effect (problem for vaccine). Not only will a vaccine have to be able to destroy the virus, it will also have to be able to destroy infected cells. Gp41 is the fusogen. Syncytia are most often seen in brain.

Reverse transcription and integration 

This is similar to other retroviruses. HIV uses reverse transcriptase imported during infection as part of the virus. The nucleocapsid enters the cytoplasm and reverse transcription occurs within the nucleocapsid. In naive resting T4 cells, the provirus (DNA form) remains in the cytoplasm, possibly because of the lack of ATP necessary for the energy-dependent import of the pre-integration complex into the nucleus. Most viruses that replicate in the nucleus can do so only in dividing cells but cell division is not essential for HIV replication. This is because two viral proteins (Vpr and one of the GAG proteins) have nuclear localization signals and so nuclear membrane breakdown at mitosis, which allows penetration of viral DNA to the chromosomes, is not necessary. 

Integration

After uncoating and entry into the nucleus, both linear and circular forms of the viral DNA are found. Linear double strand viral DNA is inserted into the host cell chromosomes using the viral integrase protein (translated from the pol gene). After integration, viral RNA is transcribed by host RNA polymerase II.
 

More on RNA transcription

Assembly of the virus occurs at the surface membrane of the cell

The virus buds and the protease cuts itself free of the POL polyprotein

Further proteolytic cleavage occurs and the virus matures

Formation of polyproteins and their cleavage

Assembly of new virus takes place at the membrane of the host cell (figure 19). Three types of protein make up the virion. These are the membrane protein complex (Gp120 and Gp41 - originally derived from Gp160) plus two internal precursor proteins, the Gag polyprotein and the Gag/Pol polyprotein (the latter is the result of a frame shift that allows the ribosome to continue translation from the Gag gene into the Pol gene)

The proteins aggregate at the cell membrane and the membrane pinches off (figure 19 and 20). The larger internal precursor  (Gag-Pol) draws two strands of the positive strand RNA into the nascent virion and the protease (part of the Gag-Pol protein) cuts itself free. The protease completes the cleavage of Gag-Pol to liberate other enzymes (reverse transcriptase, integrase and more protease). The protease also cleaves the remainder of Gag-Pol and the smaller Gag into structural proteins. p24, p7 and p6 form the bullet-shaped core while p24 underlies the membrane. The Gag and Gag/Pol fusion proteins are made in ratio of about 20:1.

This specific viral protease is vital as the viral proteins are not functional unless separated. This specificity makes the protease a good candidate for inhibition by anti-HIV drugs (see appendix 3 and anti-viral chemotherapy). Gag/Pol and Gag are attached to the viral membrane via a fatty acid that is covalently bound. The cleavages result in p17 remaining attached to the membrane.

Gp160 is translated from a singly spliced mRNA in association with the endoplasmic reticulum and is an integral membrane protein that spans the membrane once. In the endoplasmic reticulum, it is glycosylated before being transferred to the Golgi Body where it is further glycosylated and cleaved by a host enzyme to gp120 and gp41. It moves to the cell membrane via the exocytic pathway. In contrast to Gag and Gag-Pol proteins, gp160 is not cleaved by the viral protease.

 

OTHER SECTIONS ON HIV

PART I HUMAN IMMUNODEFICIENCY VIRUS AND AIDS

PART II HIV AND AIDS, THE DISEASE

PART III COURSE OF THE DISEASE

PART IV PROGRESSION AND COFACTORS

PART V STATISTICS

PART VI  SUBTYPES AND CO-RECEPTORS

PART VII  COMPONENTS AND LIFE CYCLE OF HIV

PART VIII  LATENCY OF HIV

PART IX GENOME OF HIV

PART X  LOSS OF CD4 CELLS

PART XI   POPULATION POLYMORPHISM

APPENDIX I  ANTI-HIV VACCINES

APPENDIX II  DOES HIV CAUSE AIDS?

APPENDIX III  ANTI-HIV CHEMOTHERAPY

 

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