VPR MEDIATES CELL DEATH

Apoptotic vs. Necrotic Death

Maintenance of proper cell number requires a balance between proliferation, production, and pro­grammed cell death (PCD). Inhibition of apoptosis leads to autoimmune disease and development of malignancies.

Almost all the HIV-1 proteins were implicated in modulating the apoptotic response of T cells. However, genetic analyses that directly correlate with the apoptosis-inducing activities of these proteins are lacking and raise significant concern as to the physiological involvement of these proteins as mediators of T cell death. Vpr is not a unique protein in this regard, although an emerging pool of literature has documented naturally occurring mutations in Vpr that directly abrogate cell death both in vitro and in vivo.

Early studies on Vpr function initially found that expression of Vpr-induced myocytes to differentiate⁷⁸ and led to reports demonstrating that the Vpr’s action was mediated by the gluco­corticoid receptor complex.^79,80 Given that glucocorticoids inhibit the action of NF-κB, Vpr was proposed to modify cellular functions by inducing a glucocorticoid effect, perhaps through modu­lation of T cell death or survival. Answers to how Vpr induced cell death came from studies transfecting Vpr into cells. In these cells, Vpr potently suppressed immune activation through upregulation of IκBα, which inhibited NF-κB transcriptional activity and resulted in the induction of cell death.⁸¹ These initial results led several investigators to examine details of Vpr-induced cell death. As noted previously, Vpr induces cell-cycle arrest at the G2 stage of the cell cycle and subsequently results in apoptosis of a number of human cells, including fibroblasts, T cell lines, and primary cells.^9-11,82-85 These observations were universally validated in a number of different systems. For example, in one study, adenovirus was used to introduce Vpr into HeLa cells in the absence of other HIV gene products.¹⁰ Adenovirus infection with Vpr led to G2/M cell-cycle arrest within 24 h after infection, and staining with annexin V revealed significant apoptosis with increased activation of caspase-9. Moreover, mice expressing a transgene for Vpr on a CD4 promoter exhibited enhanced apoptotic cell death with a marked decrease in T cells of the lymphoid organs and peripheral blood.⁸⁶

The major components of apoptosis initiated via the intrinsic mitochondrial pathway were largely clarified in recent years (reviewed in Green and Reed⁸⁷ and Green and Kroemer⁸⁸).

Using synthetic Vpr peptides, it was demonstrated that a major target of Vpr is the ANT, a component of the inner mitochondrial permeability transition pore complex.⁷⁰ Vpr binding to ANT causes loss of membrane potential, release of cytochrome c, and activation of caspase-3 and caspase- 9.^{9,10,70,85,89} The ability of Vpr to induce cell death seems to be dependent on the C-terminal H(S∕F)RIG motif, as specific mutations in this region abrogate Vpr’s ability to bind ANT and also reduce the capacity to induce cell death.^11,70,89

Given the important role of the mitochondria in the production of ATP, the nucleotide pool within the cell provides an indirect measure of cellular bioenergetics. NMR analysis of α, β, and γ³¹P in HeLa cells infected with adenovirus containing Vpr demonstrated a lower density of α³¹P than that of β³¹P. When this analysis was used to compare β³¹P in necrotic cells vs. adenovirus Vpr-infected cells, necrotic cells had no detectable β^3lP, whereas adenoviral-infected cells contained significant levels (reviewed in Muthumani et al.9⁰). Because the apoptotic cascade is an energy­dependent process and necrosis is associated with energy collapse, this result is consistent with Vpr-infected cells maintaining active metabolism and cytoplasmic integrity and providing further evidence that apoptosis is the main pathway in which Vpr executes cell death.

The mechanism of HIV-l induced death was called into question based on studies demon­strating that infection of Jurkat and H9 T cell lines failed to exhibit characteristics of classical apoptosis.7³ This included assays that measured caspase activity and activation, DNA fragmenta­tion, annexin V exposure, and loss of mitochondrial membrane potential. Cells overexpressing the antiapoptotic proteins Bcl-xL or v-FLIP failed to prevent death. Furthermore, no difference in cytopathogenicity to HIV-1 infection was observed in Jurkat cells deficient for RIP, caspase-8, and FADD, or when caspase inhibitors were used. Therefore, the authors concluded that a caspase and a mitochondrial-independent mechanism were responsible for the observed cell death. In a concurrent article, the same authors used electron microscopy to distinguish apoptotic vs. necrotic cell death based on cell morphology. In this case, VSV-G pseudotype viruses were engineered to contain various HIV-1 gene products and were then used to infected T cell lines. In line with the notion that nonapoptotic pathways are induced by HIV-1, the authors found that Env was not required for HIV-1-induced T cell death.⁷⁴ The C-terminal of Vpr was found to induce cell death even in the absence of caspase-9, AIF, or Apaf-1.⁸³ Also the overexpression of Bcl-2 does not protect against Vpr-induced death, lending support to the notion that T cell death may occur through multiple pathways.

There were also several reports that Vpr may function as an antiapoptotic protein. HepG2 cells undergo apoptosis after treatment with sorbitol, but cells expressing Vpr were largely protected against these effects.⁹¹ In another study, low levels of Vpr stably expressed in Jurkat cells were shown to have an impaired capacity to undergo apoptosis by cycloheximide, TNF-α, anti-Fas, or serum starvation. Furthermore, cells transfected with antisense Vpr oligonucleotides were more sensitive to these apoptotic stimuli.⁹² In a later study, the same group found that the protective effect of Vpr could be recapitulated even when cells were infected in vitro with HIV-1, although this was observed only during early time points after infection.⁹³ At later time points, these infected cells ultimately underwent apoptosis.

Based on this evidence, Vpr may have a dual role in modulating the apoptotic response that is dependent on several factors, including the time of infection and the level of Vpr. It may be possible that low levels of Vpr early in infection results in protection from cell death that facilitates maximal viral replication, persistence, and viral survival. However, ultimately, at later time points, high levels of Vpr mediate the death of the host cell to support viral spreading and dissemination.

Vpr Polymorphisms

Given the multitude of functions attributed to Vpr, numerous studies have attempted to identify naturally occurring polymorphisms within Vpr that are associated with enhanced or attenuated Vpr function. Studies in macaques, chimpanzees, and human subjects underscore the important contri­bution of Vpr to disease progression. For example, macaques infected with Vpr-defective SIV strains do not exhibit progression to AIDS or have severely attenuated disease.^95-97 In a human HIV-1 infected patient and chimpanzees infected with nonfunctional Vpr, slower disease progression was observed.⁹⁸ Therefore, it is reasonable to suggest that disease progression could be attributed to Vpr function.

FIGURE 7.3 Mutations in Vpr domain affect its cellular activity.

high T cell count.¹³ However, reversion to wild-type Vpr resulted in a progressive decline in T cell numbers, strongly indicating that Vpr is an important determinant in viral pathogenesis and disease progression. In another study, arginine substitution at position 77 to glutamine was found at high frequency among LTNPs.⁸⁹ This mutation was also observed in a larger sample of LTNP patients identified through the Los Alamos database and was associated with the reduced ability of Vpr to induce T cell death in vitro and in vivo. The incidence of this mutation was independently confirmed in another report.¹⁰² However, conflicting data were recently published as part of a larger clinical study examining the clinical outcomes of patients treated with a single- vs. a double-nucleoside reverse-transcriptase inhibitor.¹⁰³ In this report, the authors found no correlation between the R77Q mutation and disease severity, progression, or absolute CD4⁺ T cell counts. Therefore, the role of Vpr mutations, specifically R77Q, was questioned as a predictor of clinical outcome. Several explanations may account for the contrasting observations. First, this study was designed to assess whether HAART masks any benefit toward disease progression that may have otherwise resulted from C-terminal mutations within the Vpr, whereas the original report studied only drug-naive HIV-1- infected patients with defined nondisease progression. Second, other viral or host factors that were suggested to play unique roles in disease progression were not defined in the latter study, whereas nef was found to be intact in all enrolled individuals, and no patient that was homozygous or heterozygous for the ∆32bp mutation in the chemokine co-receptor CCR5 was included in the former study. Third, viral load information was not available in the second study and may account for the lack of protective effects of the R77Q mutation. In addition, without viral load data, it would not be possible to speculate severity of pre-existing disease before treatment. Despite these dis­crepancies, 41% of the drug naive patients had viruses containing the R77Q mutation in Vpr, a value similar to that found for nearly 300 LTNPs in the Los Alamos database but lower than the 80% cited in the initial report. It is difficult to extract from the data in the second study whether patients who experienced an increase in CD4⁺ T cell counts were also drug naive. Perhaps the most compelling evidence will require the isolation of clinical viruses from both cohorts of patients so that careful in vitro analysis can be performed to assess the virulence of these mutant Vpr-containing isolates.

Novel Strategies to Inhibit Vpr Function

The multifunctional role of Vpr in the viral life cycle and its interaction with a number of host targets allow for the possibility of designing specific inhibitors to block Vpr function. Several line studies have demonstrated robust inhibition of Vpr activity in in vitro systems, and it will be important to validate these potential agents in animal models.

Cyclophilin blockers such as cyclosporine A and NIM811 were shown to have potent antiret­roviral activity. For example, NIM811 interferes with nuclear localization of preintegration com­plexes. Cyclosporine was shown to block expression of interleukin-2, thereby exhibiting indirect antiviral activity through reducing T cell activation and release of infectious virions.¹⁰⁴ In rhesus monkeys acutely infected with SIV, cyclosporine A showed some benefit in preventing T cell loss,¹⁰⁵ but human trials in advanced stages of AIDS showed only a mild effect in patients treated with cyclosporine A. Because cyclophilin A inhibitors are less toxic, these drugs need to be evaluated clinically for and in combination with current HIV therapy.

Vpr-mediated cell-cycle arrest was observed in fission yeast systems and acts through a pathway involving PP2A, Wee1, and cdc25c.^29,106 In addition, Vpr experimental evidence demonstrates that Vpr induces cell killing of budding yeast through its effect on the mitochondrial transition pore complex.⁷⁰ Therefore, an elegant genetic system using the budding yeast S. cerevisiae was employed to screen for GST-fusion hexamic peptides, which suppressed the growth arrest phenotype of HIV-1 Vpr.¹⁰⁷ In this system, yeast expression plasmids were generated, encoding Vpr under the control of galactose-inducible GAL1 promoter. When grown in Vpr-inducible medium, cells transformed with wild-type Vpr underwent growth arrest compared with control. When a hexamic peptide library consisting of 1 ? 108 GST peptides was transformed into yeast, 15 clones were found to exhibit strong growth defects. Sequence analysis revealed that all GST-inhibiting peptides contained a conserved diW motif (WXXF) and a stretch of hydrophobic residues at their C-terminals. This WXXF motif was previously shown to interact with Vpr,¹⁰⁸ but the basis of this interaction is not known. Furthermore, these peptides interfered with the ability of VSV-G pseudotype viruses to induce G2 arrest and apoptosis. It was concluded that these diW-containing peptides may be capable of preventing protein-protein interactions between Vpr and host targets or may act as dissociative inhibitors. Along similar lines, other investigators examined the effects of dominant negative Vpr mutants on HIV-1 replication. Mutation of arginine residue 73 to serine in Vpr reduced the trans­activation and G2 arrest by wild-type Vpr.^109,110 However, the ability of this dominant negative R73S to block apoptosis was not investigated. Other approaches were to use antagonists of the glucocor­ticoid receptor (RU486) and pentoxifylline.⁷⁹

Using another similar yeast genetic approach, one group screened for suppressors of G2 arrest (reviewed in Bukrinsky and Zhao¹¹¹). Out of more than 2.0 ? 10⁴ transformants, two small heat­shock proteins, Hsp27 and Hsp70, were identified to inhibit G2 arrest by Vpr. When transfected into 293T cells, Hsp27 strongly suppressed G2 arrest induced by VSV-G pseudotype HIV-1 infection, whereas HSP70 had similar suppressive properties. The ability of HIV-1 to replicate in nonprolif­erating macrophages is dependent on the function of Vpr. It is believed that Vpr assists in the translocation of viral PICs via the nuclear envelope through interactions with a number of host proteins, including nucleoporins, importins, and heat-shock proteins. Hsp70 was found to stimulate nuclear import in a cell-free system.¹¹² More recently, Hsp70 expression was induced following HIV-1 infection of macrophages.¹¹³ Interestingly, in the absence of Vpr, Hsp70 was able to stimulate import and replication in macrophages. However, Hsp70 reduced replication and reversed G2 arrest in Vpr- sufficient viruses. The authors concluded that Hsp70 and Vpr act in parallel pathways when expressed separately but inhibit each other’s activities when dually expressed. These results may provide an opportunity to modulate Hsp70 function during HIV-1 infection.

Recent work showed that the use of RNAi technology may have potential in inhibiting HIV-1 replication and other viral functions. Small interfering RNAs (siRNAs) are RNA duplexes of 21 to 23 nucleotides that induce specific degradation of a homologous mRNA that results in gene siliencing.^114-116 One of the earliest findings that HIV-1 infection could be inhibited was the discovery that homozymous mutations in the chemokine co-receptors CCR5 conferred protection against HIV-1 infection.¹¹⁷ At least one other study showed that knockdown of CCR5 provided strong protection against infection in vitro,¹¹⁸ another potential target of RNAi during the viral uncoating and entry process of the target cell.

Another approach was to develop a system to deliver exogenous siRNAs through endogenous expression via DNA plasmids. This method would bypass the need to produce siRNA in cells, which have low expression of Dicer.^119,120 Still, the requirement for stem-loop structure processing by Dicer or another endonuclease was problematic. Recent work showed that these stem-loop structures can be processed in a manner similar to micro-RNA precursors, and anti-HIV siRNAs processing from micro-RNA precursors can occur efficiently.¹²¹ Both HIV-1 Rev and Tat can be inhibited by these approaches in both transient transfection or from a lentiviral transduction of hematopoietic progenitor cells.^122,123 In these studies, cotransfection of siRNA directed against Rev and Tat reduced the level of HIV-1 NL4-3 proviral DNA and inhibition of p24 expression by 4 logs¹²³ in human T cell lines and primary lymphocytes.¹²⁴ In similar work, silencing of CD4 blocked viral entry and syncytia formation.¹²⁵ Given the proof of concept in these studies, it will be of interest to determine whether HIV-1 Vpr may be a potential target for siRNA silencing. Conceiv­ably, the knockdown of Vpr expression would prevent infection of nondividing cells as well as abrogate its cell-cycle-inducing functions. Also, decreased Vpr expression may mimic naturally occurring mutations in Vpr, whereby these alleles have reduced capacity to induce cell death of target cells.

The advances in siRNA technology and continued improvement in its efficiency provide a unique opportunity to exploit delivery of anti-HIV treatments, including those that target Vpr. Loss- of-function Vpr studies indicate that inhibition of Vpr activity on cell cycle, apoptosis, or nuclear integration may be an alternative strategy to curb the deleterious effects of HIV-1 infection in humans.

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