VIRAL MEDIATORS OF DIRECT, NONAPOPTOTIC CELL DEATH
A major unresolved issue in AIDS pathogenesis is the role of individual viral proteins in HIV-1- induced necrosis. Cell death has been attributed to most HIV-1 genes, including Env, Tat, Nef, Vpr, and Vpu.
However, certain studies have also described antiapoptotic properties for nearly all of them, with the exception of Env.75,76 Functions of HIV-1 proteins in infected cells are well documented in most cases, and cellular factors interacting with them are being uncovered. Nevertheless, a confusing and contradictory picture emerges regarding their roles in death-signal initiation and its mechanism.Env
Env is one of the best-studied HIV-1 proteins (discussed in Chapter 6). Various groups have reported that the cross-linking of CD4 by gp120 primes cells for apoptosis and that mutated Env decreases cytopathicity.6,29,75,76 At the same time, other studies have demonstrated that Env-induced death does not require cellular apoptotic factors and results in necrosis.6,77,78 In studying the effect of Env on cell death, complications arise from the fact that Env is directly involved in virus entry. Any modification in Env could decrease virus infectivity and indirectly reduce viral killing. To circumvent this problem, recent studies have used VSV-G pseudotyped viruses to investigate the requirement of Env in necrosis.11,44 The results revealed no difference between Env and Env+ strains of HIV-Inl43 in their ability to cause direct killing by necrosis in a single round of infection. These data indicate that Env is dispensable for the virally induced necrosis. It should be noted that the deletion study shows only that Env is not required for HIV-1 killing and does not exclude the possibility that Env could participate in cell death.
Viral Genome, Regulatory Proteins
A regulatory protein, Tat, is suggested to be a causative agent of apoptosis; however, antiapoptotic activity has also been documented (details discussed in Chapter 9).
Intriguingly, studies with HIV-1 strains carrying mutated Tat also indicate that Tat is not required for direct necrosis during acute infection.79-81 This conclusion has been underscored by a different experiment using a derivative of an NL4-3 strain, NL-EGFP, that carries mutations in the genome so that it expresses only Tat, Rev, and Vpu as well as GFP inserted in the viral genome.12 The NL-EGFP virus pseudotyped with VSV-G was highly infectious and replicated in a Jurkat T cell line. However, unlike the wild-type counterpart, it did not cause cytopathicity during an acute single round of infection. Therefore, Tat, Rev, and Vpu, as well as the HIV-1 RNA genome, are not death-inducing factors in this system.Accessory Genes
Nef
The investigation of viral genetic variations among putative long-term nonprogressors and AIDS patients revealed Nef gene deletions in nonprogressors.82-84 Moreover, Nef can upregulate an apoptosis-inducing receptor, Fas, and its ligand, FasL (discussed in Chapter 8),75,76 although other studies excluded their involvement in CD4+ T cell destruction, as described above. The role of Nef in CD4+ T cell destruction was rendered uncertain by the data that CD4+ T cell death induced by HIV-1 infection is independent of Fas or apoptosis.6,7,10-12 Moreover, according to deletion studies, Nef-deficient strains of NL4-3 and HXB2 retain a potent ability to kill infected cells.11,12,85,86 These data suggest that Nef is also dispensable for HIV-1-induced cell death during an acute single round of infection.
Vpr
Vpr is a well-studied accessory protein that is conserved among primate lentiviruses (details discussed in Chapter 7). It is primarily found in the nucleus. In addition to its effect on mitochondria, Vpr arrests infected cells at the G2/M phase of cell cycle, as discussed above. The cell-cycle arrest by Vpr was suggested to correlate with its ability to kill infected T cells.65,71,72,74 Vpr is proposed to upregulate provirus expression by causing G2/M cell-cycle arrest, which could indirectly contribute to HIV-1 killing.
Studies with Vpr mutants demonstrated that varying degrees of cell-cycle arrest by Vpr induce a proportional amount of cell death relative to arrest,65 supporting the popular hypothesis that cell-cycle arrest contributes to Vpr killing.67,72,74 However, other studies demonstrated that the addition of drugs that abrogate the G2/M-phase arrest, including methylxanthines and pentoxifyline, did not prevent Vpr-induced cell death.65,70,73,87,88 In addition, several Vpr mutants or a GFP fusion abrogate cell-cycle arrest but still maintain their ability to cause cell death,70,73,89 raising a possibility that cell-cycle arrest may not be the cause of Vpr-mediated cell death. Further clarification is needed to establish a causative relationship between virally induced cell-cycle arrest and HIV-1 killing. It will be especially important to understand how Vpr causes cell cycle arrest and necrotic death from the nucleus, its presumed locus of action.Vpu
The small accessory protein Vpu is a membrane protein involved in the release of virions from infected cells.90-92 Its suggested function also includes the induction of CD4 degradation.93-96 The requirement of Vpu in HIV-1 killing is controversial due to conflicting reports. Vpu is implicated in HIV-1-induced apoptosis in CD4+ T cells by its ability to inhibit NF-κB activity and, consequently, the expression of antiapoptotic factors, including Bcl-xL. However, several groups reported that viral killing of CD4+ T cells was not altered with mutant HIV-1 strains, NL4-3∆Vpr∆Vpu∆Nef, HXB-2, and HIV-EGFP, which lack Vpu as well as Vpr and Nef expression.12,85,86 Therefore, at the present time, it is uncertain as to whether Vpu is necessary for HIV-1 killing.