Dr Richard Hunt

VIDEO LECTURE

VIROLOGY - CHAPTER   SEVEN    

PART NINE

HUMAN IMMUNODEFICIENCY VIRUS AND AIDS  

The Genome of HIV

LINKS  TO OTHER HIV AND AIDS SECTIONS ARE AT THE BOTTOM OF THIS PAGE

 

Figure  24A The genome of HIV-1

Figure 24B The structure of gp160.
A. The linear domain structure of gp160 is shown at the top. Gp160 is cleaved into gp120 (the surface protein) and gp41 (the transmembrane fusion protein).
B. A trimer of gp120/gp41 is associated with the viral membrane.
C. Gp120 has a number of hypervariable domains (V1-V5). The red bars show disulfide bridges

|Since HIV has a more complex life cycle that simple retroviruses such as RSV and it appears that HIV can control its replication in a more complex fashion, we might expect more genetic information but this is not so. The HIV genome is 9749 nucleotides - about the same size as other retroviruses, for example Rous sarcoma virus (RSV).

The genome of HIV is more complex than RSV, however, since it has extra open reading frames that clearly code for small proteins (figure 24a). Antibodies against these small proteins are found in HIV-infected people. Some of these are protein synthesis-controlling proteins.

The HIV genome has nine open reading frames (leading to nine primary translation products) but 15 proteins are made in all as a result of cleavage of three of the primary products.

The GAG gene and the GAG and POL genes together are translated into large polyproteins which are then cleaved by a virus-encoded protease that is part of the POL polyprotein. 

GAG polyprotein  is cleaved to into four proteins that are found in the mature virus:  MA (matrix), CA (capsid), NC (nucleocapsid), p6

POL polyprotein is cleaved to: PR (protease), RT (reverse transcriptase), IN (integrase)

ENV gene is translated to a polyprotein (Gp160) which is then cleaved by a host cell protease (called furin) that is found in the Golgi Body. It is not cleaved by the virus-encoded protease. Gp160 is cleaved to: SU (Gp120) and TM (Gp41). The latter retains the transmembrane part of Gp160 while Gp120 remains attached to Gp41 via non-covalent bonds.

In a addition to the nine proteins derived from  GAG, POL and ENV, there are six other proteins made by HIV. Three of these are incorporated into the virus (Vif,  Vpr and Nef), while the others are not found in the mature virus: Tat and Rev are regulatory proteins and Vpu indirectly assists in assembly. The genes that encode these proteins are known by three letter names that are derived as follows:

TAT: Trans-Activator of Transcription

REV: Regulator of Virion protein expression

NEF: Negative Regulatory Factor

VIF: Virion Infectivity Factor

VPU: Viral Protein U

VPR: Viral Protein R

These genes encode small proteins; TAT for example consists of 88 amino acids. They overlap with the structural genes (especially ENV) but are in different reading frames. From the above diagram of the organization of the HIV genome (figure 24), it can be seen  that some are encoded in two exons (unlike the structural genes) and therefore their mRNAs can be derived by alternative splicing of structural gene mRNAs. This is rather important to the way in which the levels of these are controlled. Mutants in the TAT and REV genes show that both proteins are necessary for virus production.

TAT

TAT gene product binds to a sequence in all of the genes of HIV and positively stimulates transcription. It is thus a positive regulator of protein synthesis, including its own synthesis.

REV

REV binds to an element only in the mRNA for structural proteins (GAG/POL/ENV) and regulates the ratio of GAG/POL/ENV to non-structural, controlling protein (TAT/REV) synthesis. When REV levels are high, structural protein synthesis rises and controlling protein synthesis falls. Thus, REV inhibits its own production and that of TAT.

The normal result is homeostasis, low or non-existent virus production and latency in the resting CD4 cell.

There is an inherent problem in HIV's lifestyle. It uses genomic RNA as its messenger RNA. This RNA is unspliced and the nucleus has a mechanism to prevent unspliced mRNAs from leaving the nucleus and being translated. It is the function of Rev to overcome this problem.

Figure 25  NEF effects on CD4

Figure 26   NEF induces cytokines that attract T cells to an infected macrophage

NEF

Nef protein is synthesized early in infection. Despite its small size NEF has several functions.

a) Homeostasis leads to problems for the parasitic provirus: 

i) Super-infection by other HIV particles which bind to surface CD4 antigen of the infected cell may kill the cell. 
ii) Probably more importantly, virus bound via CD4 antigen at the cell surface or free Gp120 bound to CD4 antigen at the cell surface may result in the cell being subject to an immune attack and the infected cell may be destroyed.

The translation of the NEF gene as a result of the first infecting virus causes the internalization of CD4 antigen from the cell surface and its destruction in lysosomes (figure 25). Thus no more HIV or gp120 can bind to the surface of an infected cell! 

b) By a different mechanism from its down regulation of CD4 antigen, NEF reduces surface expression of MHC class I molecules. This alters antigen presentation by the infected cell and is proposed to protect the infected cell from attack by cytotoxic T cells

c) The name, NEF, comes from negative factor. Originally, it seemed that virions that lacked NEF grew better than wild type. Now the consensus is for the opposite, that is that virus produced in the presence of NEF is a little more infectious than virus produced in its absence.

d) It is found that NEF is important for HIV replication in vivo but there seems to be much less effect of NEF in an in vitro cell culture situation. Why this is so has long been obscure. Recently, this question seems to have been solved. The answer is found in the macrophages which are changed in two ways when they are infected with a NEF-expressing HIV (remember that macrophages are the cells that bring HIV into the body and the initial strains of HIV in an infected patient are macrophage-tropic).  

HIV-infected macrophages secrete MIP-1alpha and MIP-1beta. These are two chemokines that  bind to the co-receptors for HIV infection of macrophages but here these chemokines have another  function.  

They cause resting CD4+ T cells to migrate (undergo chemotaxis) towards the infected macrophages (figure 26). This is important in vivo since, initially, HIV-infected cells are not very numerous and uninfected T cells may not be in the vicinity of the infected cells. Moreover, HIV does not have a very long half life in the circulation before becoming non-infectious. Migration of uninfected cells towards infected cells increases the probability that the T cells will encounter infected macrophages before they leave the reticuloendothelial system. This explains why NEF seems not to be of much consequence in cell culture where the cells are already close together. The infected macrophages do something else. They make a factor that has not yet been identified that activates the resting T cells that have been attracted towards them allowing the T cells to be productively infected and to shed new virus (Remember, lentiviruses, unlike most other retroviruses can infect non-dividing cells. Normally, retroviruses can only enter the nucleus and integrate during mitosis when the nuclear membrane is broken down; however, proteins of lentviruses such as HIV have nuclear targeting signals that allow the nucleocapsid to find the nuclear pore. Thus, the virus can integrate into the host cell chromosome where it can now code for more viral RNA and protein. HIV, unlike some other lentiviruses, does not transcribe its genome to RNA in resting T cells since the activation of the promotor in the LTR requires transcription factors that are only made when a resting T cell becomes activated).  These finding explain why macrophages are vital for the spread of HIV.

Note: In vivo, HIV can infect a resting T cell but cannot replicate in that cell. Although NEF can activate the cell, it cannot be made in the resting T cell. The above observations solve this conundrum. Macrophages are infected by HIV and make NEF without any activation process. As  a result they make factors that activate resting T cells that can now support a productive infection!

VPU 

After activation of the T cell, the virus faces another problem: CD4 antigen and Gp120 are being made in the endoplasmic reticulum of the same cell. They are likely to bind to one another before reaching the plasma membrane and such complexes are usually targeted by the cell for degradation. To stop this unfortunate state of affairs, another of the small HIV proteins (VPU) promotes the proteolysis of the CD4 antigen of the host cell as it is made!

VPU also enhances viral particle release from the host cell. How it does this is not clear but VPU forms an ion channel in the plasma membrane of the host cell and may alter the ionic composition of the cytoplasm. It also binds to a cellular protein (Vpu-binding protein or UBP) and over-expression of this protein diminishes the enhancing effect of VPU on virus release. UBP may be a negative factor for assembly that must be displaced from one of the GAG proteins before virus can assemble at the cell surface.

VPU forms an ion transporting pore in the surface membrane that conducts small ions such as Na and K. The ability of VPU to form channels and its ability to enhance viral release seem to correlate.

From its ability to stimulate viral release and also to break down CD4 antigen (which are separate functions of different parts of the VPU molecule), it appears that VPU enhances the pathogenicity of the virus by increasing the number of HIV particles per cell.

VIF 

Vif (viral infectivity factor) protein, which is essential for infection in vivo, may be very important in suppressing resistance to HIV infection by the host. Vif is needed during late stages of virus production and seems to function by suppressing innate anti-viral activities in T cells and macrophages, the major cells that are infected in humans. Without Vif, HIV is not infectious in primary human T cells. 
What is it about the T cells that render them active against HIV when Vif is absent? It is thought that Vif is needed for production of infectious virus because it inhibits an antiviral pathway in the cells that involves an enzyme called APOBEC3G (originally discovered as an apolipoprotein B messenger RNA editing enzyme). This enzyme is a cytidine deaminase that also targets single stranded DNA. It appears that Vif prevents the editing of the single strand form of the viral DNA that is the initial product of reverse transcription. Such editing is much more pronounced when the infecting virus has a Vif deletion. Thus, Vif prevents many mutations that would lead to changes in the structural proteins, enzymes and regulatory proteins that would otherwise result in loss of viral infectivity.

VPR

VPR influences the pathogenesis of HIV and is essential for infection of macrophages, and to a lesser extent of other cells. It also activates HIV LTR-promoted transcription. It causes the arrest of host cell division in the G2 stage of the cell cycle and apoptosis of the infected cell. It acts as a cytoplasmic-nucleus shuttle protein (for the pre-integration complex through the nuclear pores). VPR is found in the serum of HIV-infected patients.

WEB RESOURCES

Humans have anti-HIV gene

PART I HUMAN IMMUNODEFICIENCY VIRUS AND AIDS

PART II HIV AND AIDS, THE DISEASE

PART III COURSE OF THE DISEASE

PART IV 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  OTHER CELLS INFECTED BY HIV AND POPULATION POLYMORPHISM

APPENDIX I  ANTI-HIV VACCINES

APPENDIX II  DOES HIV CAUSE AIDS?

APPENDIX III  ANTI-HIV CHEMOTHERAPY