Grouppe Kurosawa: HIV - The Biology of VPR (print)

The Biology of VPR

VPR is a viral protein that is specific for primate lentiviruses. The feline immunodeficiency virus (FIV) lacks a VPR gene, which may be the reason some infected cats can live with the virus without symptoms. Simian immunodeficiency virus (SIV) VPR deletion mutants still cause AIDS in rhesus monkeys, but the severity of the disease is attenuated. Monkeys infected with VPR mutants live longer and have stronger humoral and cell-mediated immune responses than animals infected with wild-type viruses. In tissue culture, NEF and VPR mutants do not impair the replication of SIV. However, in rhesus monkeys both NEF and VPR mutants readily revert to wild-type and this reversion is positively correlated with the development of disease. Since low virus burdens and no disease have been reported in VPR and NEF mutants which did NOT revert to wild-type, there is apparently a strong selective pressure for functional forms of VPR and NEF in HIV infections.

When peripheral blood cells are removed from HIV-infected donors and cultured in vitro, the cells do not secrete virus unless activated by antigens, mitogens or cytokines. Latent infections are the rule not the exception during the asymptomatic period of AIDS. Although TNF is well known to stimulate viral production, we believe this only occurs during the symptomatic period of disease. TNF secretion is probably suppressed by glucosteroids, VPR, and TGFb and this contributes to the long duration of the asymptomatic period in Western countries. HIV-1 virions contain up to 1000 VPR proteins. These proteins are released from disintegrating virions (half-life two days); virally infected cells have the ability to activate viral production from latently infected cell lines and peripheral blood mononuclear cells, possibly by their release of VPR and TAT into the extracellular environment. VPR is able to induce HIV expression in a manner analogous to pro-inflammatory cytokines and provides a means by which viral particles, whether infectious or not, can regulate viral production in an autocrine or paracrine fashion. In a lymphoid organ, high concentrations of extracellular VPR could regulate the life cycle of HIV infectivity, while serum antibodies regulate the amount of free VPR in the blood. A breakdown in the production of high affinity antibodies against VPR, such as by a polyclonal non-specific activation of B cells by NEF, TAT, and the immune hormones TGFb and IL-10, may lead to increasing amounts of free, non-immune complexed VPR in the blood and, subsequently, increased viral titers. The ability of VPR to regulate latency answers a number of perplexing questions about the relationship between viral titers and pathogenesis. It is known that the amount of viral RNA in the blood directly correlates with disease severity. However, what is not fully appreciated is that over 99% of the virus found in blood is non-infectious or dead. The total number of virions in the blood exceed infectious units by factors of 10,000 to 10,000,000. These are very impressive numbers and they strongly suggest that dead, non-infectious viral particles play a role in the pathogenesis of AIDS.

The biology of VPR has not been as extensively studied as other viral proteins, such as TAT. However, enough is known about this protein to strongly warrant its inclusion in an HIV vaccine. VPR has different effects on monocytes and T cells. VPR prevents primary infected T cells from becoming chronically infected viral reservoirs. Only T cell lines, adapted to tissue culture, can chronically produce virus. VPR has a cytolytic effect on T cell growth by inhibiting p34cdc2 activity and arresting cells in G2 of the cell cycle. Prolonged G2 blockage invariably induces apoptosis, and death of the newly infected cells. The maintenance of HIV infections in T cells appears to be regulated by two competing processes: the rate at which virions can spread and establish new infections and the rate at which previously infected cells disappear. These are exactly the conclusions adopted by the Ho and Shaw groups who found that protease inhibitors caused a rapid decrease in viral titers and a rebound in the number of CD4 T cells in the blood. Since VPR's effects on T cell growth are independent of the presence of virus, VPR may play a significant role in controlling the cell cycle dynamics of non-infected T cells. For example, in the presence of protease inhibitors, the viral/VPR titer in the blood will decrease. As previously secreted VPR is cleared from the blood and tissues, uninfected T cells, blocked in their cell cycle by VPR, will reenter the cell cycle and proliferate. This could partially account for the rapid increase in CD4 T cells reported by Ho after the initiation of protease inhibitor therapy. Another explanation for the sudden increase in CD4 cells may have nothing to do with cellular kinetics. HIV viral protease inhibitors also inhibit the chymotrypsin activity of the proteasome complex. Since a number of different viral proteins induce the degradation of CD4 molecules in the proteasome, the inhibition of the proteasome may have the reverse effect-increased numbers of CD4 molecules will accumulate in the membranes of infected and non-infected cells.

VPR has quite different effects on viral production in mononuclear phagocytes. In macrophages, an important reservoir for viral production, VPR is required for the efficient infection and replication of virus. Macrophages are non-proliferating cells and VPR promotes the nuclear localization of viral nucleic acids in non-dividing cells. T cells do not require VPR in order to integrate the provirus into their DNA The loss of VPR reduces viral production in macrophages by up to 1000 fold.

Glucosteroids share many of the same immunosuppressive properties of Cyclosporin. In fact, calcineurin activation protects T cells from glucosteroid-induced apoptosis, or programmed cell death, while glucosteroids inhibit IL-2 gene transcription by a calcineurin-dependent pathway. In addition to its effects on calcineurin, glucosteroid-receptor complexes directly interact with transcription factors such as AP-1, CREB and NF-kB, thereby physically blocking their ability to bind DNA. Glucosteroids also suppress immunity by stimulating the synthesis of a protein called IkBa, an inhibitor of the transcription factor NF-kB. NF-kB plays a major role in activating many target genes of relevance to AIDS. They include:

o Cytomegalovirus
o HIV1/2
o Adenovirus
o Immunoglobulin kappa light chain
o T cell receptor alpha/beta chains
o MHC class I/II antigens
o Beta-2 microglobulin
o Endothelial leukocyte adhesion molecule-1 (ELAM-])
o Vascular cell adhesion molecule-1 (VCAM-])
o Intercellular cell adhesion molecule-1 (ICAM- 1)
o Beta interferon
o GM-CSF
o G-CSF
o M-CSF
o IL-2
o IL-6
o IL-8
o TNF alpha/beta
o C-rel
o NF-kB precursor p105
o C-myc
o Nitric oxide synthese

If VPR and other viral proteins activate the glucosteroid receptor, they will induce an immunosuppresslon mediated primarily, but not exclusively, by inhibition of NF-kB responsive genes. Although there is a GRE (glucosteroid response element) at position +5002 within the VIF open reading frame, there is no evidence that glucosteroids administered in vivo increase the viral titer. In fact, quite the reverse is true. Dexamethasone, a synthetic steroid, inhibits HIV LTR-directed gene expression in T cells, but not in macrophages. Prednisolone, another synthetic glucosteroid, significantly reduced serum levels of p24gag after 4 weeks of therapy. In addition, and most significantly, synthetic glucosteroids administered to HIV infected individuals slow the loss of CD4 cells and inhibit antigen-induced apoptosis of the same cell population. Dexamethasone inhibits the ability of macrophages to delete CD4 cells via anti-CD4 antibody or immune complexed HIV envelope protein gp120. The deletion of normal CD4 cells by macrophages was also inhibited by dexamethasone. Upregulation of CD95 expression on T cells by anti-CD4 or gp120/IgG complexes, predisposing factors in the induction of apoptosis, is inhibited by dexamethasone in a dose dependent manner. The in vivo evidence is quite clear that prolonged prophylactic therapy with glucosteroids either inhibit or have no effect on HIV viral titers. The prolonged asymptomatic period of AIDS may be strongly related to the ability of VPR to block the transcription of NF-kB dependent genes and to induce the activation of glucosteroid dependent genes. In Africa, the average HIV infected person dies within 3 years. Africans have high levels of nitric oxide, and TNFa in their blood due to persistent infections with malaria, TB, and parasites. TNFa and NO are well established activators of HIV gene transcription. In Western countries, the asymptomatic period of infection alone may exceed 10 years in the absence of drug therapy. People in Western countries are not exposed to the disease, malnutrition, and overall stress in living experienced by the world's poor. All these factors contribute to a rapid disease progression. When the viral titer and secretion of pro-inflammatory hormones reach a certain level, the tissues of the body become resistant to the effects of endogenously secreted glucosteroids (similar to how asthmatics become resistant to inhaled glucosteroids over time). When glucosteroid resistance becomes pronounced, all the normal physiological feedback restraints in place against the excessive secretion of pro-inflammatory, HIV inducing immune hormones are no longer functional. The development of wide spread glucosteroid resistance in most of the tissues of the body is the single most important physiological event that PERMITS actual clinical AIDS to occur. In the absence of glucosteroid resistance, you are left with a troublesome chronic viral infection that is NOT fatal.