Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest.

The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.     

The regulation of primate immunodeficiency virus infectivity by Vif is cell species restricted: a role for Vif in determining virus host range and cross-species transmission.

The primate immunodeficiency virus Vif proteins are essential for replication in appropriate cultured cell systems and, presumably, for the establishment of productive infections in vivo. We describe experiments that define patterns of complementation between human and simian immunodeficiency virus (HIV and SIV) Vif proteins and address the determinants that underlie functional specificity. Using human cells as virus producers, it was found that the HIV-1 Vif protein could modulate the infectivity of HIV-1 itself, HIV-2 and SIV isolated from African green monkeys (SIVAGM). In contrast, the Vif proteins of SIVAGM and SIV isolated from Sykes' monkeys (SIVSYK) were inactive for all HIV and SIV substrates in human cells even though, at least for the SIVAGM protein, robust activity could be demonstrated in cognate African green monkey cells. These observations suggest that species-specific interactions between Vif and virus-producing cells, as opposed to between Vif and virus components, may govern the functional consequences of Vif expression in terms of inducing virion infectivity. The finding that the replication of murine leukemia virus could also be stimulated by HIV-1 Vif expression in human cells further supported this notion. We speculate that species restrictions to Vif function may have contributed to primate immunodeficiency virus zoonosis.     

Human and simian immunodeficiency virus-mediated upregulation of the apoptotic factor TRAIL occurs in antigen-presenting cells from AIDS-susceptible but not from AIDS-resistant species

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections lead to AIDS in humans and rhesus macaques (RM), while they are asymptomatic in species naturally infected with SIV, such as chimpanzees, sooty mangabeys (SM), and African green monkeys (AGM). Differential CD4(+) T-cell apoptosis may be responsible for these species-specific differences in susceptibility to disease. To identify factors that influence the different apoptotic responses of these species, we analyzed virus-infected human and nonhuman primate peripheral blood mononuclear cells (PBMC). We found that the apoptotic factor TRAIL was present at higher levels in human and RM PBMC cultures and was mediating, at least in part, CD4(+) T-cell apoptosis in these cultures. The species-specific increase in TRAIL and death receptor expression observed with cultures also occurred in vivo in SIV-infected RM but not in SIV-infected SM. In human and RM myeloid immature dendritic cells and macrophages, the virus-induced expression of TRAIL and other interferon-inducible genes, which did not occur in the same cells from chimpanzee, SM, and AGM, was Tat dependent. Our results link the differential induction of TRAIL in human and nonhuman primate cells to species-specific differences in disease susceptibility.     

Human immunodeficiency virus type 1 Vpr interacts with HHR23A, a cellular protein implicated in nucleotide excision DNA repair.

The human immunodeficiency virus type 1 (HIV-1) vpr gene is an evolutionarily conserved gene among the primate lentiviruses HIV-1, HIV-2, and simian immunodeficiency viruses. One of the unique functions attributed to the vpr gene product is the arrest of cells in the G2 phase of the cell cycle. Here we demonstrate that Vpr interacts physically with HHR23A, one member of an evolutionarily conserved gene family involved in nucleotide excision repair. Interaction of Vpr with HHR23A was initially identified through a yeast two-hybrid screen and was confirmed by the demonstration of direct binding between bacterially expressed recombinant and transiently expressed or chemically synthesized protein products. Visualization of HHR23A and Vpr by indirect immunofluorescence and confocal microscopy indicates that the two proteins colocalize at or about the nuclear membrane. We also map the Vpr-binding domain in HHR23A to a C-terminal 45-amino-acid region of the protein previously shown to have homology to members of the ubiquitination pathway. Overexpression of HHR23A and a truncated derivative which includes the Vpr-binding domain results in a partial alleviation of the G2 arrest induced by Vpr, suggesting that the interaction between Vpr and HHR23A is critical for cell cycle arrest induced by Vpr. These results provide further support for the hypothesis that Vpr interferes with the normal function of a protein or proteins involved in the DNA repair process and, thus, in the transmission of signals that allow cells to transit from the G2 to the M phase of the cell cycle.     

Vpr-induced cell cycle arrest is conserved among primate lentiviruses.

We previously reported that expression of human immunodeficiency virus type 1 strain NL4-3 (HIV-1(NL4-3))vpr causes cells to arrest in the G2 phase of the cell cycle. We examined the induction of cell cycle arrest by other HIV-1 isolates and by primary lentiviruses other than HIV-1. We demonstrate that the vpr genes from tissue culture-adapted or primary isolates of HIV-1 are capable of inducing G2 arrest. In addition, we demonstrate that induction of cell cycle arrest is a conserved function of members of two other groups of primate lentiviruses, HIV-2/simian immunodeficiency virus strain sm (SIVsm)/SIVmac and SIVagm. vpr from HIV-1, HIV-2, and SIVmac induced cell cycle arrest when transfected in human (HeLa) and monkey (CV-1) cells. vpx from HIV-2 and SIVmac did not induce detectable cell cycle arrest in either cell type, and SIVagm vpx was capable of inducing arrest in CV-1 but not HeLa cells. These results indicate that induction of cell cycle perturbation is a general property of lentiviruses that infect primates. The conservation of this viral function throughout evolution suggests that it plays a key role in virus-host relationships, and elucidation of its mechanism may reveal important clues about pathology induced by primary lentiviruses.     

A Novel Simian Immunodeficiency Virus (SIVdrl) pol Sequence from the Drill Monkey, Mandrillus leucophaeus

The drill monkey has been shown by serology and PCR to harbor a unique simian immunodeficiency virus (SIVdrl). A pol sequence, amplified from uncultured peripheral blood cells, is most closely related to the equivalent SIV sequences from the red-capped mangabey (SIVrcm), the sabaeus African green monkey (SIVagmSAB), and the chimpanzee (SIVcpz) and to the human immunodeficiency virus type 1 (HIV-1) sequence of humans. It is as yet unclear whether SIVdrl has a mosaic genome like SIVrcm and SIVagmSAB, is a member of the SIVcpz/HIV-1 lineage, or represents a novel primate lentivirus lineage.     

Retrovirus restriction by TRIM5alpha variants from Old World and New World primates

The TRIM5alpha proteins of humans and some Old World monkeys have been shown to block infection of particular retroviruses following virus entry into the host cell. Infection of most New World monkey cells by the simian immunodeficiency virus of macaques (SIVmac) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5alpha orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5alpha proteins functionally resembled human TRIM5alpha, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5alpha proteins from several New World monkey species restricted infection by SIVmac and the SIV of African green monkeys, SIVagm. Spider monkey TRIM5alpha, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIVmac, SIVagm, HIV-1, and N-MLV. Tandem duplications in the TRIM5alpha B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5alpha proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5alpha proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.     

Species-specific, postentry barriers to primate immunodeficiency virus infection

By using replication-defective vectors derived from human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV(mac)), and murine leukemia virus (MuLV), all of which were pseudotyped with the vesicular stomatitis virus (VSV) G glycoprotein, the efficiency of postentry, early infection events was examined in target cells of several mammalian species. Titers of HIV-1 vectors were significantly lower than those of SIV(mac) and MuLV vectors in most cell lines and primary cells from Old World monkeys. By contrast, most New World monkey cells exhibited much lower titers for the SIV(mac) vector compared with those of the HIV-1 vector. Prosimian cells were resistant to both HIV-1 and SIV(mac) vectors, although the MuLV vector was able to infect these cells. Cells from other mammalian species were roughly equivalent in susceptibility to the three vectors, with the exception of rabbit cells, which were specifically resistant to the HIV-1 vector. The level of HIV-1 vector expression was very low in transduced cells of rodent, rabbit, cow, and pig origin. Early postentry restriction of primate immunodeficiency virus infection exhibits patterns largely coincident with species borders and applies to diverse cell types within an individual host, suggesting the involvement of species-specific, widely expressed cellular factors.     

A conserved dileucine-containing motif in p6(gag) governs the particle association of Vpx and Vpr of simian immunodeficiency viruses SIV(mac) and SIV(agm)

Vpr is a small accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is specifically incorporated into virions. Members of the HIV-2/SIV(sm)/SIV(mac) lineage of primate lentiviruses also incorporate a related protein designated Vpx. We previously identified a highly conserved L-X-X-L-F sequence near the C terminus of the p6 domain of the Gag precursor as the major virion association motif for HIV-1 Vpr. In the present study, we show that a different leucine-containing motif (D-X-A-X-X-L-L) in the N-terminal half of p6(gag) is required for the incorporation of SIV(mac) Vpx. Similarly, the uptake of SIV(mac) Vpr depended primarily on the D-X-A-X-X-L-L motif. SIV(mac) Vpr was unstable when expressed alone, but its intracellular steady-state levels increased significantly in the presence of wild-type Gag or of the proteasome inhibitor lactacystin. Collectively, our results indicate that the interaction with the Gag precursor via the D-X-A-X-X-L-L motif diverts SIV(mac) Vpr away from the proteasome-degradative pathway. While absent from HIV-1 p6(gag), the D-X-A-X-X-L-L motif is conserved in both the HIV-2/SIV(sm)/SIV(mac) and SIV(agm) lineages of primate lentiviruses. We found that the incorporation of SIV(agm) Vpr, like that of SIV(mac) Vpx, is absolutely dependent on the D-X-A-X-X-L-L motif, while the L-X-X-L-F motif used by HIV-1 Vpr is dispensable. The similar requirements for the incorporation of SIV(mac) Vpx and SIV(agm) Vpr provide support for their proposed common ancestry.     

Differential apoptosis effects of primate lentiviral Vpr and Vpx in mammalian cells

The growth inhibitory effects of Vpr and Vpx are species- and cell type-dependent. HIV-1, HIV-2 and SIV Vpr are primarily cytostatic in mammalian cells and HIV-1 Vpr has been reported to induce apoptosis in human cells. Our previous studies have shown that HIV-1, HIV-2 and SIV Vpr and Vpx have differential cytostatic and cytotoxic effects in the yeast cells [Zhang et al.: Virology, 230:103-112; 1997]. Here, we further examined the apoptosis function of HIV-1 Vpr in different species of mammalian cells and investigated if other primate lentiviral Vpr and Vpx exert similar functions. Our results show that none of the primate lentiviral Vpr or Vpx we tested induces apoptosis in nonhuman species of mammalian cells. However, HIV-1 Vpr, but not HIV-2 or SIV Vpr and/or Vpx, induced apoptosis in different types of human cell lines. Further, the apoptotic effect of HIV-1 Vpr can be distinguished from that of the human interferon-gamma, a known proapoptotic protein, that HIV-1 Vpr shows little to no paracrine and/or bystander effect. When coexpressed with Bcl-2 or Bcl-X(L), the apoptotic effect of HIV-1 Vpr became markedly attenuated. These results indicate that the apoptotic effect of HIV-1 Vpr is species-dependent and is intracellularly modulated by the Bcl-2 family of proteins. Our study also suggests that the proapoptotic function of HIV-1 Vpr is developmentally associated with human but not nonhuman primate species.     

Human immunodeficiency virus type 1 Vpr modifies cell proliferation via multiple pathways.

Vpr, one of the accessory molecules of HIV-1, has been demonstrated to arrest the cell cycle at the G2 phase. This Vpr-mediated cell cycle arrest is implicated to have an important role in the viral life cycle. In the present study, we quantitate the extent of Vpr-mediated cell cycle arrest with the use of a bicistronic vector consisting of a vpr gene and a green fluorescence protein sequence. Using this system, we examined the effect of several Vprs on cell cycle progression and growth of cells from different species quantitatively. We found that Vpr from the T-cell line-adapted HIV-1SF2 strain (Vpr2) could not significantly induce G2 arrest in HeLa cells but was able to induce it in 293T cells. However, strong inhibition of cell proliferation in HeLa cells as well as in 293T cells was observed by Vpr2. This ability of Vpr2 to inhibit cell proliferation without G2 arrest was also observed when expressed in monkey cell line. Analyses of chimeric Vprs revealed that this species-non-specific growth inhibitory activity of Vpr was not mediated solely by the C-terminal region of Vpr. These results indicated that the growth inhibitory activity of Vpr is independent of its G2 arresting activity. In addition, the species-non-specific nature of this activity suggests that Vpr has a novel mechanism to retard cell proliferation by influencing basic cellular functions.     

Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction

Primate lentiviruses encode four "accessory proteins" including Vif, Vpu, Nef, and Vpr/Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. We present evidence that the Vpx proteins of HIV-2/SIV(SM) promote virus infection by antagonizing an antiviral restriction in macrophages. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in trans overcame the restriction to HIV-1 and SIV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. Our results indicate that macrophage harbor a potent antiviral restriction and that primate lentiviruses have evolved Vpx to counteract this restriction.     

Genetic studies with the fission yeast Schizosaccharomyces pombe suggest involvement of wee1, ppa2, and rad24 in induction of cell cycle arrest by human immunodeficiency virus type 1 Vpr

Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G(2)/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast Schizosaccharomyces pombe, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (cdc phenotype) typical of G(2)/M cell cycle arrest in wild-type and various mutant strains of S. pombe was examined. Our results indicated that Vpr caused the cdc phenotype in wild-type S. pombe as well as in strains carrying mutations, such as the cdc2-3w, Deltacdc25, rad1-1, Deltachk1, Deltamik1, and Deltappa1 strains. However, other mutants, such as the cdc2-1w, Deltawee1, Deltappa2, and Deltarad24 strains, failed to show a distinct cdc phenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the cdc phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.     

The level of CD4 expression limits infection of primary rhesus monkey macrophages by a T-tropic simian immunodeficiency virus and macrophagetropic human immunodeficiency viruses

The entry of primate immunodeficiency viruses into cells is dependent on the interaction of the viral envelope glycoproteins with receptors, CD4, and specific members of the chemokine receptor family. Although in many cases the tropism of these viruses is explained by the qualitative pattern of coreceptor expression, several instances have been observed where the expression of a coreceptor on the cell surface is not sufficient to allow infection by a virus that successfully utilizes the coreceptor in a different context. For example, both the T-tropic simian immunodeficiency virus (SIV) SIVmac239 and the macrophagetropic (M-tropic) SIVmac316 can utilize CD4 and CCR5 as coreceptors, and both viruses can infect primary T lymphocytes, yet only SIVmac316 can efficiently infect CCR5-expressing primary macrophages from rhesus monkeys. Likewise, M-tropic strains of human immunodeficiency virus type 1 (HIV-1) do not infect primary rhesus monkey macrophages efficiently. Here we show that the basis of this restriction is the low level of CD4 on the surface of these cells. Overexpression of human or rhesus monkey CD4 in primary rhesus monkey macrophages allowed infection by both T-tropic and M-tropic SIV and by primary M-tropic HIV-1. By contrast, CCR5 overexpression did not specifically compensate for the inefficient infection of primary monkey macrophages by T-tropic SIV or M-tropic HIV-1. Apparently, the limited ability of these viruses to utilize a low density of CD4 for target cell entry accounts for the restriction of these viruses in primary rhesus monkey macrophages.     

A challenge to the ancient origin of SIVagm based on African green monkey mitochondrial genomes

While the circumstances surrounding the origin and spread of HIV are becoming clearer, the particulars of the origin of simian immunodeficiency virus (SIV) are still unknown. Specifically, the age of SIV, whether it is an ancient or recent infection, has not been resolved. Although many instances of cross-species transmission of SIV have been documented, the similarity between the African green monkey (AGM) and SIVagm phylogenies has long been held as suggestive of ancient codivergence between SIVs and their primate hosts. Here, we present well-resolved phylogenies based on full-length AGM mitochondrial genomes and seven previously published SIVagm genomes; these allowed us to perform the first rigorous phylogenetic test to our knowledge of the hypothesis that SIVagm codiverged with the AGMs. Using the Shimodaira-Hasegawa test, we show that the AGM mitochondrial genomes and SIVagm did not evolve along the same topology. Furthermore, we demonstrate that the SIVagm topology can be explained by a pattern of west-to-east transmission of the virus across existing AGM geographic ranges. Using a relaxed molecular clock, we also provide a date for the most recent common ancestor of the AGMs at approximately 3 million years ago. This study substantially weakens the theory of ancient SIV infection followed by codivergence with its primate hosts.