Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes

All primate lentiviruses known to date contain one or two open reading frames with homology to the human immunodeficiency virus type 1 (HIV-1) vpr gene. HIV-1 vpr encodes a 96-amino-acid protein with multiple functions in the viral life cycle. These functions include modulation of the viral replication kinetics, transactivation of the long terminal repeat, participation in the nuclear import of preintegration complexes, induction of G2 arrest, and induction of apoptosis. The simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagm) contains a vpr homologue, which encodes a 118-amino-acid protein. SIVagm vpr is structurally and functionally related to HIV-1 vpr. The present study focuses on how three specific functions (transactivation, induction of G2 arrest, and induction of apoptosis) are related to one another at a functional level, for HIV-1 and SIVagm vpr. While our study supports previous reports demonstrating a causal relationship between induction of G2 arrest and transactivation for HIV-1 vpr, we demonstrate that the same is not true for SIVagm vpr. Transactivation by SIVagm vpr is independent of cell cycle perturbation. In addition, we show that induction of G2 arrest is necessary for the induction of apoptosis by HIV-1 vpr but that the induction of apoptosis by SIVagm vpr is cell cycle independent. Finally, while SIVagm vpr retains its transactivation function in human cells, it is unable to induce G2 arrest or apoptosis in such cells, suggesting that the cytopathic effects of SIVagm vpr are species specific. Taken together, our results suggest that while the multiple functions of vpr are conserved between HIV-1 and SIVagm, the mechanisms leading to the execution of such functions are divergent.     

Evolution of the primate lentiviruses: evidence from vpx and vpr.

The genomes of the four primate lentiviral groups are complex and contain several regulatory or accessory genes. Two of these genes, vpr and vpx, are found in various combinations within the four groups and encode proteins whose functions have yet to be elucidated. Comparison of the encoded protein sequences suggests that the vpx gene within the HIV-2 group arose by the duplication of an ancestral vpr gene within this group. Evolutionary distance analysis showed that both genes were well conserved when compared with viral regulatory genes, and indicated that the duplication occurred at approximately the same time as the HIV-2 group and the other primate lentivirus groups diverged from a common ancestor. Furthermore, although the SIVagm vpx proteins are homologous to the HIV-2 group vpx proteins, there are insufficient grounds from sequence analysis for classifying them as vpx proteins. Because of their similarity to the vpr proteins of other groups, we suggest reclassifying the SIVagm vpx gene as a vpr gene. This creates a simpler and more uniform picture of the genomic organization of the primate lentiviruses and allows the genomic organization of their common precursor to be defined; it probably contained five accessory genes: tat, rev, vif, nef and vpr.     

Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells.

We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.     

Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells

Mutants of human immunodeficiency virus type 2 (HIV-2) carrying a frame-shift mutation in vpx, vpr, and in both genes were monitored for their growth potentials in a newly established lymphocytic cell line, HSC-F. Worthy of note, the replication of a vpx single mutant, but not vpr, was severely impaired in these cells, and that of a vpx-vpr double mutant was more damaged. Defective replication sites of the vpx single and vpx-vpr double mutants were demonstrated to be mapped, respectively, to the nuclear import of viral genome, and to both, this process and the virus assembly/release stage. While the mutational effect of vpr was small, the replication efficiency in one cycle of the vpx mutant relative to that of wild-type virus was estimated to be 10%. The growth phenotypes of the vpx, vpr, and vpx-vpr mutant viruses in HSC-F cells were essentially repeated in primary human lymphocytes. In primary human macrophages, whereas the vpx and vpx-vpr mutants did not grow at all, the vpr mutant grew equally as well as the wild-type virus. These results strongly suggested that Vpx is critical for up-regulation of HIV-2 replication in natural target cells by enhancing the genome nuclear import, and that Vpr promotes HIV-2 replication somewhat, at least in lymphocytic cells, at a very late replication phase.     

Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics.     

Characterization of three nef-defective human immunodeficiency virus type 1 strains associated with long-term nonprogression. Australian Long-Term Nonprogressor Study Group

Long-term survivors (LTS) of human immunodeficiency virus type 1 (HIV-1) infection provide an opportunity to investigate both viral and host factors that influence the rate of disease progression. We have identified three HIV-1-infected individuals in Australia who have been infected for over 11 years with viruses that contain deletions in the nef and nef-long terminal repeat (nef/LTR) overlap regions. These viruses differ from each other and from other nef-defective strains of HIV-1 previously identified in Australia. One individual, LTS 3, is infected with a virus containing a nef gene with a deletion of 29 bp from the nef/LTR overlap region, resulting in a truncated Nef open reading frame. In addition to the Nef defect, only viruses containing truncated Vif open reading frames of 37 or 69 amino acids could be detected in peripheral blood mononuclear cells isolated from this patient. LTS 3 had a viral load of less than 20 copies of RNA/ml of plasma. The other two long-term survivors, LTS 9 and LTS 11, had loads of less than 200 copies of RNA/ml of plasma and are infected with viruses with larger deletions in both the nef alone and nef/LTR overlap regions. These viruses contain wild-type vif, vpu, and vpr accessory genes. All three strains of virus had envelope sequences characteristic of macrophagetropic viruses. These findings further indicate the reduced pathogenic potential of nef-defective viruses.     

The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection.

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that can cause extensive cytopathicity in T cells. However, long-term productive infection of T-cell lines has been described. Here we show that although Vpr has no effect on the initial cytopathic effect of HIV-1, viruses that contain an intact vpr gene are unable to establish a chronic infection of T cells. However, virus with a mutated vpr gene can readily establish such long-term cultures. The effect of Vpr is independent of the env gene and the nef gene. Furthermore, expression of Vpr alone affects the progression of cells in the cell cycle. These results suggest that HIV-1 has evolved a viral gene to prevent chronic infection of T cells.     

Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection.

The matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein contains a highly basic region near its amino terminus. It has been proposed that this basic domain, in conjunction with the HIV-1 accessory protein Vpr, is responsible for the localization of the HIV-1 preintegration complex to the nucleus in nondividing cells. It has also been postulated that the matrix basic domain assists in the targeting of the HIV-1 Gag precursor Pr55Gag to the plasma membrane during virus assembly. To evaluate the role of this highly basic sequence during infection of primary human monocyte-derived macrophages, single- and double-amino-acid-substitution mutations were introduced, and the effects on virus particle production, Gag protein processing, envelope glycoprotein incorporation into virus particles, and virus infectivity in the CEM(12D-7) T-cell line, peripheral blood mononuclear cells, and primary human monocyte-derived macrophages were analyzed. Although modest effects on virus particle production were observed with some of the mutants, none abolished infectivity in primary human monocyte-derived macrophages. In contrast with previously reported studies involving some of the same matrix basic domain mutants, infectivity in monocyte-derived macrophages was retained even when combined with a vpr mutation.     

Open reading frame vpr of simian immunodeficiency virus encodes a virion-associated protein.

The genomes of simian immunodeficiency viruses isolated from rhesus macaques (SIVmac) contain an open reading frame (ORF), vpr, which has a coding potential of 97 to 101 amino acid residues. In this study, a vpr ORF-encoded protein of approximately 11 kDa was identified, and anti-vpr antibodies were detected in rhesus macaques infected by SIVmac. These results provide clear evidence that the vpr ORF is a coding gene of SIVmac. The vpr protein, like the vpx protein which is encoded by another accessory gene of SIVmac, was also found to be associated with viral particles. This observation demonstrates that more than one accessory gene product can be present in the virions of this family of retroviruses and raises the possibility that the vpr protein may have a role in early part of the virus life 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.     

Replication and pathogenicity of human immunodeficiency virus type 1 accessory gene mutants in SCID-hu mice.

The functional roles of the human immunodeficiency virus type 1 (HIV-1) accessory genes (nef, vpr, vpu, and vif) are as yet unclear. Using the SCID-hu model system, we have examined the infectivity, replicative capacity, and pathogenicity of strains of the molecular clone HIV-1NL4-3 that contain deletion mutations in these individual accessory genes. We determined that deletion of these genes had differential effects on both infectivity and pathogenicity. Deletion of vpr had little or no effect on viral infectivity, replication, and pathogenicity; however, deletion of vpu or vif had a significant effect on infectivity and moderate effects on pathogenicity. nef-minus strains were the most attenuated in this system, demonstrating significantly lower levels of infectivity and pathogenicity. However, deletion of these individual genes attenuated but did not abrogate the pathogenic properties of HIV-1. Mutant viruses still retained the ability to induce thymocyte depletion to various degrees if implants were infected with higher doses of virus or observed for longer periods of time. The relative contributions of these genes to in vivo pathogenic potential should be taken into consideration when one is contemplating a live attenuated vaccine for HIV-1.     

Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.     

Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.     

Mutational analysis of the human immunodeficiency virus vpr open reading frame.

Mutations were introduced by recombinant DNA techniques into the vpr open reading frame of an infectious molecular clone of human immunodeficiency virus type 1. The effect of these changes on the replicative and cytopathologic properties of the virus recovered from transfected cells was studied in several human CD4+ lymphocyte cell lines. In all cases, mutant viruses were infectious and cytopathic. However, when a low-input dose was used, mutants grew significantly more slowly than the wild-type virus. The growth kinetics of vpr mutants were distinct from those of vif and vpu mutants.     

Antisense phosphorothioate oligodeoxynucleotides targeted to the vpr gene inhibit human immunodeficiency virus type 1 replication in primary human macrophages.

The replication of human immunodeficiency viruses (HIV) in human macrophages is influenced by genetic determinants which have been mapped predominantly to the viral envelope. However, in HIV-2, the vpr gene has also been suggested as an important modulator of viral expression in human macrophages. We synthesized five antisense phosphorothioate oligodeoxynucleotides complementary to the vpr mRNA of HIV-1Ba-L, a highly macrophage-tropic viral strain, and measured their effect on HIV-1Ba-L replication in primary human macrophages. All of the oligodeoxynucleotides displayed some level of non-sequence-specific inhibition of viral replication; however, only the antisense one had an additional effect on viral production in primary macrophages. Of the five antisense oligodeoxynucleotides tested, only one did not show any additional effect on viral production, whereas all the others inhibited viral replication to a similar degree (70 to 100%). Variation in the degree of inhibition was observed by using five different donors of human primary macrophages. The phosphorothioate oligonucleotides, targeted to the initiating methionine of the Vpr protein, had an inhibitory effect at both 20 and 10 microM only when the size was increased from 24 to 27 bases. Thus, HIV-1 replication in human macrophages is modulated by the expression of the vpr gene, and it is conceivable that vpr antisense oligodeoxynucleotides could be used in combination with antisense oligodeoxynucleotides against other HIV-1 regulatory genes to better control viral expression in human macrophages.     

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.     

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.     

Effect of reciprocal complementation of two defective human immunodeficiency virus type 1 (HIV-1) molecular clones on HIV-1 cell tropism and virulence.

Human immunodeficiency virus type 1 (HIV-1) displays both interstrain and intrastrain genetic variability. Virus populations with extensive microheterogeneity have been defined as swarms or quasispecies. Many of the genomes within HIV-1 swarms appear to be defective in one or more genes required for viral replication. It is unclear to what extent defective viruses play a role in the process of HIV-1 infection or in the pathogenesis of AIDS. We have isolated two biologically active HIV-1 clones: LW 12.3, which contains defects in the vif and vpr genes, and MN ST.1, which has a defect in the vpu gene. LW 12.3 is unable to replicate in peripheral blood mononuclear cells (PBMC). The growth of MN-ST.1 in SupT1 cells is marked by a 3-week lag in extracellular virus production and by the presence of unusually abundant viral buds. We demonstrate here that coinfection of PBMC with these two partially defective HIV-1 clones extends the cellular host range of LW 12.3, significantly increases the replication rate of both viral genomes, and eliminates the delay in production observed with the vpu-defective MN ST.1. When the lesions in vpr and vif of LW 12.3 are repaired, the resultant virus grows normally in PBMC. This is also the case when only vif is repaired, indicating that complementation of LW 12.3 in PBMC by MN ST.1 is mediated by vif in trans. The reciprocal complementation results in a dramatic increase of HIV-1 virulence. This two-component model represents a simplified version of the in vivo situation and illustrates one way in which interaction of defective viruses could increase the spread of infection and progression of disease.     

Simian immunodeficiency virus from African green monkeys.

Simian immunodeficiency virus (SIV) was isolated from the total peripheral blood mononuclear cell population and the monocyte-macrophage adherent cell population of three seropositive green monkeys originating from Kenya. SIV from these African green monkeys (SIVagm) was isolated and continuously produced with the MOLT-4 clone 8 (M4C18) cell line but not with a variety of other cells including HUT-78, H9, CEM, MT-4, U937, and uncloned MOLT-4 cells. Once isolated, these SIVagm isolates were found to replicate efficiently in M4C18, SupT1, MT-4, U937, and Jurkat-T cells but much less efficiently if at all in HUT-78, H9, CEM, and MOLT-4 cells. The range of CD4+ cells fully permissive for replication of these SIVagm isolates thus differs markedly from that of previous SIV isolates from macaques (SIVmac). These SIVagm isolates had a morphogenesis and morphology like that of human immunodeficiency virus (HIV) and other SIV isolates. Antigens of SIVagm and SIVmac cross-reacted by comparative enzyme-linked immunosorbent assay only with reduced efficiency, and optimal results were obtained when homologous antibody and antigen were used. Western blotting (immunoblotting) of purified preparations of SIVagm isolate 385 (SIVagm385) revealed major viral proteins of 120, 27, and 16 kilodaltons (kDa). The presumed major core protein of 27 kDa cross-reacted antigenically with the corresponding proteins of SIVmac (28 kDa) and HIV-1 (24 kDa) by Western blotting. Hirt supernatant replicative-intermediate DNA prepared from cells freshly infected with SIVagm hybridized to SIVmac and HIV-2 DNA probes. Detection of cross-hybridizing DNA sequences, however, required very low stringency, and the restriction endonuclease fragmentation patterns of SIVagm were not similar to those of SIVmac and HIV-2. The nucleotide sequence of a portion of the pol gene of SIVagm385 revealed amino acid identities of 65% with SIVmac142, 64% with HIV-2ROD, and 56% with HIV-1BRU; SIVagm385 is thus related to but distinct from previously described primate lentiviruses SIVmac, HIV-1, and HIV-2. Precise information on the genetic makeup of these and other SIV isolates will possibly lead to better understanding of the history and evolution of these viruses and may provide insight into the origin of viruses that cause acquired immunodeficiency syndrome in humans.