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HIV-1 groups M, N, O, and P resulted from four independent zoonotic transmission events of simian immunodeficiency viruses (SIV) infecting chimpanzees and gorillas to humans. Studying the differences between pandemic HIV-1 group M and closely related but less successful pathogens helps to expose viral vulnerabilities, which can be exploited as targets of new treatment or preventive strategies. One characteristic that distinguishes HIV-1 group M strains from the geographically-limited and less prevalent groups N, O and P is the acquisition of adaptive mutations in the accessory protein Vpu that mediate potent activity against human tetherin. This adaptation is thought to be critical for the effective spread of HIV-1 M strains within the human population because human tetherin harbours a deletion that renders it resistant to the counteraction by its SIV precursor. However, direct evidence supporting this hypothesis has been very limited. To address this issue and determine the contribution of Vpu-mediated tetherin counteraction to viral fitness, I mutated the AxxxA transmembrane domain Vpu motif of primary HIV-1 group M and N strains to specifically disrupt their ability to antagonize tetherin, but not other Vpu functions, such as downmodulation of CD4, CD1d and NTB-A, and suppression of NF-��B activity. Reversion of this particular human-specific adaptation significantly reduced the ability of HIV-1 group M Vpu proteins to enhance virus production and release from primary CD4+ T cells in the presence of type I interferon (IFN) by about 2- to 5-fold. These consistent differences between wild type and tetherin-defective mutant HIV-1 group M viruses highlight the important role of Vpu-mediated tetherin antagonism in viral release and resistance to IFN. Surprisingly, transmitted founder (TF) HIV-1 strains exhibited higher virion release capacity than chronic control HIV-1 strains irrespective of Vpu function. In addition, all pandemic group M viruses produced higher levels of cell-free virions than the poorly-adapted HIV-1 N group strain. In agreement with these in vitro data showing that Vpu-mediated tetherin antagonism plays an important role in HIV-1 fitness, the anti-tetherin activity was found to be critical for an efficient early spread of HIV-1 in humanized mice as shown in a study performed by our collaborator. Thus, efficient virus release from infected cells seems to play an important role in the spread of pandemic HIV-1 and requires a Vpu protein that efficiently counteracts human tetherin.

Objective:Identifying the genetic variability in vertically transmitted viruses in early infancy is important to understand the disease progression. Being important in HIV-1 disease pathogenesis, vpu gene, isolated from young infants was investigated to understand the viral characteristics.Method:Blood samples were obtained from 80 HIV-1 positive infants, categorized in two age groups; acute (6-18 months). A total of 77 PCR positive samples, amplified for vpu gene, were sequenced and analyzed.Results:73 isolates belonged to subtype C. Analysis of heterogeneity of amino acid sequences in infant groups showed that in the sequences of acute age group both insertions and deletions were present while in the early age group only deletions were present. In the acute age group, a deletion of 3 residues (RAE) in the first alfa helix in one sequence and insertions of 1-2 residues (DM, GH, G and H) in the second alfa helix in 4 sequences were observed. In the early age group, deletion of 2 residues (VN) in the cytoplasmic tail region in 2 sequences was observed. Length of the amino terminal was observed to be gradually increasing with the increasing age of the infants. Protein Variation Effect Analyzer software showed that deleterious mutations were more in the acute than the early age group. Entropy analysis revealed that heterogeneity of the residues was comparatively higher in the sequences of acute than the early age group.Conclusion:Mutations observed in the helixes may affect the conformation and lose the ability to degrade CD4 receptors. Heterogeneity was decreasing with the increasing ages of the infants, indicating positive selection for robust virion survival.

For a long time it was thought that human tetherin is resistant to lentiviral Nef proteins due to a five amino acid deletion in its cytoplasmic tail. Using FACS analysis and virus release assays, I show that Nef proteins of HIV-1 group O decrease surface levels of human tetherin and increase virus release from CD4+ T cells. Thus, Nef proteins of HIV-1 group O have adapted to be effective antagonists of human tetherin and human tetherin is not generally resistant to Nef proteins. Furthermore, I describe one strain of HIV-1 group O that uses both its Nef and Vpu protein to antagonize human tetherin, which demonstrates the importance of tetherin antagonism and the enormous plasticity of lentiviral accessory proteins.

For nonenveloped viruses such as Simian Virus 40, the mechanism used to translocate viral components across membranes is poorly understood. Previous results indicated that the minor structural proteins, VP2 and VP3, might act as membrane proteins during infection. Here, purified VP2 and VP3 were found to form pores in host cell membranes. To identify possible membrane domains, individual hydrophobic domains from VP2 and VP3 were expressed in a model protein and tested for their ability to integrate into membranes. Several domains from the late proteins supported endoplasmic reticulum membrane insertion as transmembrane domains. Mutations in VP2 and VP3 were engineered that inhibited membrane insertion and pore formation. When these mutations were introduced into the viral genome, viral propagation was inhibited. This comprehensive approach revealed that the viroporin activity of VP2 and VP3 was inhibited by targeted disruptions of individual hydrophobic domains and the loss of membrane disruption activity impaired viral infection.

Coevolution of primate lentiviruses and their host species is fueled by a continuous arms race, in which the host’s immune system induces an antiviral cellular state after sensing of the pathogen, whereas the virus evades recognition or directly counteracts antiviral effector proteins. One of the best-characterized antiretroviral restriction factors is tetherin, which inhibits the release of newly formed progeny virions by physically retaining them at the plasma membrane of the infected cell. After cross-species transmission of simian immunodeficiency viruses (SIVs) to humans, pandemic HIV-1 group M stains switched from Nef- to Vpu-mediated tetherin antagonism. We recently found that viruses from group O, the second most common group of HIV-1, are also able to counteract tetherin. These viruses have evolved their Nef protein to target a conserved region adjacent to a protective deletion that renders human tetherin resistant to SIV Nefs. In contrast, viruses from the rare HIV-1 groups N and P lack efficient tetherin antagonists, suggesting that counteraction of tetherin is a prerequisite for successful viral spread. Analysis of rare non-synonymous polymorphisms in the human tetherin gene, showed that none of them affects the ability of tetherin to restrict virus release or its sensitivity to lentiviral antagonists. However, tetherin also acts as a pattern recognition receptor inducing an antiviral immune response via activation of NF-κB. Interestingly, one of the naturally occurring polymorphisms (R19H) located in the cytoplasmic tail of tetherin abrogated its ability to act as an innate sensor. It is conceivable that group M Vpus and group O Nefs inhibit innate sensing by antagonizing tetherin. We could demonstrate, however, that lentiviral Vpu proteins also directly suppress NF-κB signaling, irrespectively of anti-tetherin activity. NF κB activation plays a complex role in lentiviral replication. On the one hand, it is required for efficient viral gene expression, while, on the other hand, it induces the expression of antiviral factors. We showed that primate lentiviruses cope with these opposing roles of NF-κB by precisely orchestrating its activation. The early protein Nef boosts NF-κB activity to initiate efficient expression of viral proteins, including Tat, which can maintain viral transcription independently of NF-κB. This allows the late protein Vpu to suppress NF-κB-mediated expression of antiviral genes without inhibiting viral gene expression. Furthermore, we found that viruses that do not encode a vpu gene, either use their Nef protein to limit T cell activation by downmodulating the CD3 T cell receptor, or directly inhibit NF-κB using their Vpr protein. The observation that primate lentiviruses have evolved at least three different strategies to inhibit NF-κB activation highlights the importance of this transcription factor in antiretroviral immunity, and illustrates the enormous functional plasticity of primate lentiviral accessory proteins. Since the activation levels of NF-κB also play a crucial role in the establishment of viral latency and reactivation, a better understanding of the regulation of NF-κB activity by primate lentiviral proteins is of high interest. Despite sophisticated mechanisms to escape innate immune sensing and to inhibit downstream signaling pathways, HIV-1 infection causes chronic immune activation resulting in the induction of numerous antiviral host restriction factors. Based on common characteristics of known restriction factors, we performed a genome-wide screen to identify novel antiviral proteins. A surprisingly large proportion of the tested factors showed antiviral properties in transient transfection assays. Guanylate-binding protein 5 (GBP5), specifically reduced the infectivity of progeny virions and was thus selected for in-depth characterization. We found that GBP5 is a key mediator of the antiviral interferon (IFN)-response that reduces the infectivity of progeny virions by interfering with the proper processing and incorporation of the viral envelope (Env) glycoprotein. Furthermore, GBP5 levels determined infectious virus yield from human macrophages, while mutations in the vpu start codon reduced the inhibitory effect of GBP5 by increasing Env expression. Our results therefore identify GBP5 as a novel antiretroviral effector of the IFN-response that is evaded by an unusual trade-off mechanism.

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated dementia (HAD) and other neurological disorders. The role of HIV regulatory and accessory proteins in the pathogenesis of these disorders is unclear. Here we analyzed sequences of tat, rev, and vpu genes in 55 subgenomic clones previously shown to encode functional env genes from brain and lymphoid tissues of four AIDS patients with HAD. Phylogenetic analysis showed distinct compartmentalization of tat, rev, and vpu genes in brain versus lymphoid tissues. Nine of 19 vpu sequences from brain of two patients had premature stop codons at positions between amino acids 2 and 30, compared with 0 of 8 from lymphoid tissues. Tat sequences from brain (n = 8 of 8) but not lymphoid (n = 0 of 6) tissue from one patient had a 35 amino acid truncation at the C-terminus. Rev sequences from the brain of one patient (n = 6 of 8) had a 5 amino acid truncation. These results demonstrate a high frequency of defective vpu compared with tat and rev genes in brain from HAD patients, and identify sequence variants of these regulatory/accessory genes that may influence the pathogenesis of HIV-associated neurological disease.

Human immunodeficiency virus type 1 (HIV) replicates only in certain primate cells. In murine cells expressing cyclin T1, a posttranscriptional block exists such that small amounts of capsid and little infectious virus are released. This block is relieved in part by fusion with human cells. Here we have tested a panel of mouse-human somatic cell hybrids for production of infectious virus. Only those containing human chromosome 2 were permissive, which correlated with capsid production. The effect was specific to HIV in that release of murine leukemia virus was minimally affected by the presence of chromosome 2. Although expression of Vpu markedly increased capsid production in the absence of chromosome 2, it did not result in a corresponding increase in infectious HIV. The presence of chromosome 2 did not have consistent effects on the amount of unspliced viral RNA, whereas the amount of cell-associated Gag p55 was increased a fewfold. These results suggest that processing of HIV Gag can be corrected by one or more genes present on human chromosome 2 to allow production of infectious HIV from murine cells.

Simian immunodeficiency viruses (SIV) naturally infect more than 30 species of African nonhuman primates, but current data suggest that the viruses identified to date represent only a fraction of nonhuman primate lentiviruses (6, 13, 24, 54). Most of the SIVs are nonpathogenic in their natural hosts (8, 51), with a few exceptions (52). This is thought to be an example of long-term cospeciation of viruses with their natural hosts (24). This view is supported by the fatal outcome of the zoonotic transmission of primate immunodeficiency viruses in humans and macaques (47). Characterization of newly identified SIV strains is necessary to gain a better understanding of the evolutionary relationships among primate lentiviruses and to identify the potential risk for new zoonotic transmissions into the human population. Cercopithecidae monkeys are divided into two subfamilies named Colobinae and Cercopithecinae. Cercopithecinae monkeys, from which most nonhuman primate lentiviruses have been isolated, are subdivided into two tribes (Cercopithecini and Papionini) and numerous species and subspecies distributed throughout sub-Saharan Africa (23). Primate lentiviruses for which full-length genome sequences are available have been classified, on the basis of phylogenetic analysis (6, 16), into six approximately equidistant major groups or lineages, as follows: (i) SIVcpz from chimpanzees (Pan troglodytes) including also human immunodeficiency virus type 1 (HIV-1) (21, 31, 45, 56, 69; (ii) SIVsm from sooty mangabey (Cercocebus atys) together with HIV-2 (10, 20, 28); (iii) SIVagm from the four major species of African green monkeys, i.e., grivet (18), sabeus (33), vervet (26), and tantalus (60) (members of the genus Chlorocebus); (iv) SIVsyk from Sykes' monkeys (Cercopithecus mitis albogularis) (27) together with SIVmon from mona monkeys (Cercopithecus mona) (3, 13), SIVgsn from greater spot-nosed guenons (Cercopithecus nictitans) (16), SIVmus from mustached monkeys (Cercopithecus cephus) (13), and SIVdeb from De Brazza's monkeys (Cercopithecus neglectus) (6); (v) the SIVlhoest lineage, composed of SIVlhoest from L'Hoest monkeys (Cercopithecus lhoesti) (25), SIVsun from sun-tailed monkeys (Cercopithecus solatus) (4), and SIVmnd-1 from mandrills (Mandrillus sphinx) (68); and (vi) SIVcol from black and white colobus (Colobus guereza) (15). Several other viruses, notably SIVrcm from red-capped mangabeys (5), SIVmnd-2 from mandrills (61), and SIVdrl from drills (29) showed discordant phylogenetic clustering in different genomic regions, and are therefore considered recombinants (29, 61, 64). Partial sequences are available for SIVtal from talapoin monkeys (Miopithecus talapoin) (50) and for SIVschm from red-tailed guenon (Cercopithecus ascanius schmidti) (70); partial sequences from two species of western colobus are available: SIVwrc from western red colobus (Piliocolobus badius) and SIVolc from olive colobus (Procolobus verus) (14). Serological surveys indicate that other species may also harbor lentiviruses, among them Allen's swamp monkey (Allenopithecus nigrovidis), Diana monkey (Cercopithecus diana), blue monkey (Cercopithecus mitis), and Hamlyn's monkey (Cercopithecus hamlyni) (24, 39, 48). However, such serologic findings need to be confirmed by molecular studies and virus characterization. For some SIVs, there is good evidence for cospeciation with the host, such as those infecting African green monkeys (1, 33, 44) and the C. lhoesti supergroup (4), whereas some others originated through cross-species transmission from other species. The best known example is that of the human viruses HIV-1 and HIV-2, which originated following cross-species transmission of SIVcpz from chimpanzees and SIVsm from sooty mangabeys, respectively (58). Classification of SIVrcm (5), SIVmnd-2 (61, 64), SIVagm.sab (33), and SIVdrl (29) is complicated because their genomes represent mosaic genomes derived from two or more distinct viral species through prior recombination events. A recent study even suggested that all six phylogenetic lineages might have arisen by recombination (55). All primate lentivirus lineages share the gag, pol, vif, vpr, tat, rev, env, and nef genes but differ by the presence or absence of the vpu/vpx accessory gene in their genome. Viruses of the SIVcpz/HIV-1 lineage, together with SIVgsn, SIVmon, and SIVmus, harbor a vpu gene, while viruses of the SIVsm/HIV-2 lineage, together with SIVrcm, SIVmnd-2, and SIVdrl, harbor a vpx gene; on the other hand, other SIVs harbor neither of the these genes. In 1999, we launched a lentivirus seroprevalence survey of wild-born monkeys in the Democratic Republic of the Congo (DRC). Here we describe the genetic characterization of a new primate lentivirus (SIVden) harbored by a Cercopithecus denti, a species belonging to the mona subgroup. Six mona species have been described (from west to east: Campbell's monkey, Lowe’s monkey, Mona monkey, crowned monkey, Wolf’s monkey, and Dent’s monkey), together with a few subspecies. Their habitat covers a large region of Africa, from Senegal to Uganda. Two independent sequences from C. mona have been characterized (3, 13), and Wolf's mona was found many years ago to be seropositive for SIV (46). SIVden belongs to the SIVsyk lineage but is genetically distinct from previously characterized primate lentiviruses. Analysis of the entire SIVden genome revealed the presence of a vpu open reading frame. Phylogenetic analyses revealed that the SIVden sequence belongs to the SIVsyk group of viruses and, more specifically, is more closely related to SIVdeb than to viruses belonging to the SIVgsn sublineage, which harbor a vpu gene.

We have constructed reading frames for the HIV-1 YU-2 minor proteins Vpr, Vpu, Vif and Nef that are codon-optimized for high-level expression in mammalian cells. We show that, in the absence of the Rev/Rev-response element system, these codon-optimized reading frames result in greatly increased levels of expression of the corresponding proteins in cell culture systems when compared with the native reading frame. Northern blot analysis shows that the increase in expression found with the codon-optimized reading frames is largely owing to increased steady-state mRNA levels.

The Vpu protein is the smallest of the proteins encoded by human immunodeficiency virus type 1 (HIV-1). This transmembrane protein interacts with the CD4 molecule in the rough endoplasmic reticulum (RER), resulting in its degradation via the proteasome pathway. Vpu also has been shown to enhance virion release from infected cells. While much has been learned about the function of Vpu in cell culture systems, its exact role in HIV-1 pathogenesis is still unknown. This has been primarily due to the lack of a suitable primate model system since vpu is found only in HIV-1 and simian immunodeficiency viruses isolated from chimpanzees (SIVcpz), and three species of old world monkeys within the genus Cercopithecus. Several laboratories have developed pathogenic molecular clones of simian-human immunodeficiency virus (SHIV) in which the tat, rev, vpu and env genes of HIV-1 are expressed in the genetic background of SIV. The availability of such clones has allowed investigators to assess the role of Vpu in pathogenesis using a relevant animal model. This review will focus on the current understanding of the structure-function relationships of Vpu protein and recent advances using the SHIV model to assess the role of Vpu in HIV-1 pathogenesis.

Human immunodeficiency virus has evolved several redundant mechanisms to remove its receptor, the CD4 molecule, from the cell surface. Indeed, HIV-1 encodes three proteins, Nef, Vpu and Env, that have a profound effect on CD4 trafficking and catabolism. Given this functional convergence, it is believed that cell surface CD4 regulation constitutes an important determinant of viral replication and pathogenesis in vivo. This review highlights recent progress made in our understanding of the molecular mechanisms underlying the down-regulation of the CD4 receptor by HIV-1 and describes our current comprehension of the role of CD4 down-regulation during HIV-1 infection.

An R5-tropic SHIV CHN19P4 was previously generated using a primary HIV- I subtype-C envelope. We have further characterized this SHIV in two species of macaques. To determine whether this isolate is transmissible vaginally, female pigtailed macaques were inoculated with 2x10 3 TCID 50 of SHIV CHN19P4 by the vaginal route. Animals became infected with a high peak plasma viremia (>10 7 viral copies/mL) and rapid seroconversion. The viremia was accompanied by CD4 + lymphocytopenia in the gut lamina propria lymphocyte (LPL) population. Comparable CD4 + T-cell loss was not seen in peripheral blood and colonic lymph nodes. These findings demonstrate a unique R5-tropic SHIV that can be used to study envelope-related issues in vaginal transmission of the most prevalent subtype of HIV- 1. We also found that rhesus macaques intravenously inoculated with I x 10 3 TCID 50 of SHIV CHN19P4 became infected and showed CD4 + lymphocytopenia in the gut LPL population. Despite inactivation of the vpu gene in SHIV CHN19P4 , the virus appears to target mainly gut-associated lymphoid tissues during the initial stage of infection as has been described for SHIV SF162P , another R5-tropic (subtype B) recombinant virus. Our data indicate that the R5-mediated CD4 + lymphocytopenia in the gut is likely independent of HIV- genotypes and of the function of vpu at the acute phase of viral infection.

Use of the macaque model of human immunodeficiency virus (HIV) pathogenesis has shown that the accessory genes nef and vpu are important in the pathogenicity of simian immunodeficiency virus (SIV) and simian–human immunodeficiency virus (SHIV). We examined the ability of two nonpathogenic SHIVs, SHIV PPC and Δ vpuΔnef SHIV PPC , to gain pathogenicity by rapid serial passage in macaques. In this study, each virus was passaged by blood intravenously four times at 4-week intervals in macaques. Animals were monitored for 40 weeks for levels of CD4 T cells and quantitative measures of virus infection. Δ vpuΔnef SHIV PPC maintained a limited phase of productive replication in the four animals, with no loss of CD4 + T cells, whereas SHIV PPC became more pathogenic in later passages, judging by plasma viral load and viral mRNA in lymph nodes, infectious peripheral blood mononuclear cells and CD4 + T cell loss. The nef, LTR, and env of the SHIV PPC viruses underwent numerous mutations, compared to Δ vpuΔnef SHIV PPC . This study confirms the seminal role that nef, LTR, and vpu could play in regulation of pathogenesis of HIV infection.

To investigate the HIV-1 subtypes prevalent in the Republic of Congo, we isolated 28 HIV-1 strains from Congolese AIDS patients in 1996 and 1997, and analyzed them phylogenetically. Phylogenetic analysis based on part of the 5' tat-env (vpu) and env sequences revealed that only 13 (46.4%) of the 28 isolates belonged to the same subtype in the vpu tree as in the env tree; the remaining 15 (53.6%) strains showed discordant subtypes between vpu and env with 6 different profiles; that is, 1 A/A (vpu/env), 1 D/D, 5 G/G, 4 H/H, 2 unclassified (U)/U, 9 G/A, 2 G/H, 1 G/J, 1 H/G, 1 U/A, and 1 U/J. Thus, 9 of the 15 discordant HIV-1s were of the G/A (vpu/env) type, and did not form any subcluster within the subtype G lineage in the vpu-based phylogenetic tree. In addition, CRF02_AG (IbNG), which is a G/A (vpu/env) type, was not found in the Republic of Congo. These data suggest that the majority of HIV-1 subtypes circulating in the Republic of Congo have mosaic structures and may have been derived from independent recombinational events.

We compared 162 vpu sequences of human immunodeficiency virus type 1 (HIV-1) in peripheral blood mononuclear cell DNA of 6 infected mother-infant pairs after perinatal transmission, and found a 90.12% frequency of intact vpu open reading frames. The heterogeneity of vpu genes between epidemiologically linked mother-infant pairs was lower compared with epidemiologically unlinked individuals. However, the variability of vpu genes was higher than that seen for other HIV-1 genes, including vif, vpr, tat, and gag p17 from the same mother-infant pairs. Moreover, the infants' sequences displayed patterns similar to those seen in their mothers. The functional domains essential for Vpu activity, including efficient release of virus particles from infected cells and CD4 degradation, were conserved in most of the sequences. In a phylogenetic analysis, the 162 sequences from 6 mother-infant pairs formed distinct clusters for each mother-infant pair sequences and grouped with subtype B sequences. These data support the importance of vpu in HIV-1 replication of mother-infant isolates that are involved in perinatal transmission.

To acquire new knowledge about the genetic diversity and potential impact on vaccine strategies of HIV-1 subtype C in South Africa, we have characterized the vif, vpr, and vpu genes of 15 isolates. Phylogenetic analysis of the genomic fragment encompassing these genes revealed subtype C subclusters, suggesting close relatedness with subtype C strains from other geographic locations and excluded isolation of South African strains. The putative T155 phosphorylation site in the C terminal of Vif was absent in all subtype C sequences. Variation in the predicted amino acid sequences of the three genes further showed strong correlation with other subtype C sequences.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein binds to the CD4 receptor and induces its degradation by cytosolic proteasomes. This process involves the recruitment of human betaTrCP (TrCP), a key member of the SkpI-Cdc53-F-box E3 ubiquitin ligase complex that specifically interacts with phosphorylated Vpu molecules. Interestingly, Vpu itself, unlike other TrCP-interacting proteins, is not targeted for degradation by proteasomes. We now report that, by virtue of its affinity for TrCP and resistance to degradation, Vpu, but not a phosphorylation mutant unable to interact with TrCP, has a dominant negative effect on TrCP function. As a consequence, expression of Vpu in HIV-infected T cells or in HeLa cells inhibited TNF-alpha-induced degradation of IkappaB-alpha. Vpu did not inhibit TNF-alpha-mediated activation of the IkappaB kinase but instead interfered with the subsequent TrCP-dependent degradation of phosphorylated IkappaB-alpha. This resulted in a pronounced reduction of NF-kappaB activity. We also observed that in cells producing Vpu-defective virus, NF-kappaB activity was significantly increased even in the absence of cytokine stimulation. However, in the presence of Vpu, this HIV-mediated NF-kappaB activation was markedly reduced. These results suggest that Vpu modulates both virus- and cytokine-induced activation of NF-kappaB in HIV-1-infected cells.

The human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that forms oligomeric structures in membranes. Expression of vpu using Sindbis virus (SV) as a vector leads to permeabilization of plasma membrane to hydrophilic molecules and impaired maturation of wild type SV glycoproteins in BHK cells. The 6K protein is a membrane protein encoded in the SV genome that facilitates budding of virus particles and regulates transport of viral glycoproteins through the secretory pathway. Some of these functions were assayed with a SV mutant containing a partially deleted 6K gene. Transfection of BHK cells with pSVΔ6K vector rendered defective SVΔ6K virus, which had lower membrane permeabilization, impaired glycoprotein processing, and deficient virion budding. Replacement of 6K function by HIV-1 Vpu in SVΔ6K was tested by cloning the vpu gene under a duplicated late promoter (pSVΔ6KVpu). The presence of the vpu gene in the 6K-deleted virus enhances membrane permeability, modifies glycoprotein precursor processing, and facilitates infectious virus particle production. Restoration of infectivity of 6K-deleted SV by Vpu was evidenced by increased PFU production and cytopathic effect on infected cells. The modification of SVΔ6K glycoprotein maturation by Vpu was reflected in augmented processing of B precursor and impairment of PE2 cleavage. Taken together, our data support the notion that HIV-1 Vpu and SV 6K proteins share some analogous functions.

We studied the replication of HIV-1 macrophage-tropic CCR5-using strains (R5) in CD4+ T cell lines to better understand the switch in co-receptor use of such strains during disease progression and to assess resulting changes in cell tropism. We found that the majority of R5 strains cannot replicate in CD4+ T cell lines without adaptation by serial passage. A small minority of primary R5 isolates, however, were able to infect two T cell lines, Molt4 and SupT1. This expanded tropism was due to the use of undetectable levels of CCR5 rather than CXCR4 or alternative receptors. In contrast, HIV-1SF162 adaptation for replication in the C8166 T cell line was due to the emergence of variant strains that could use CXCR4. Of two variants, one was dual-tropic and one T-tropic, although both could use CCR5 as well as CXCR4. A single mutation in the start codon of the accessory gene vpu accounted for the T-tropic phenotype of the second variant, indicating that a non-functional vpu impairs macrophage tropism. Thus, in vitro and in the absence of an immune response, R5 strains naturally adapt to infect CXCR4+ T cell lines. Such adaptation resembles the rare R5 to X4 switch that occurs in vivo. Mutations in accessory genes (e.g., vpu) not required for replication in rapidly dividing cell lines may also occur in vitro, abrogating replication in primary cell types such as macrophages. Such mutations, however, are normally selected against in vivo.

We compared the Vpu sequences from 101 strains of HIV-1 isolated from diverse geographical regions and various subtypes in order to identify regions of high variability, and those amino acid residues that were highly conserved or invariant. In addition to the highly conserved casein kinase II (CKII) phosphorylation sites, our analysis identified additional invariant residues in the transmembrane domain and in the first and second alpha-helical domains. Our analysis revealed that all subtype C sequences had a conserved LRLL motif at the C terminus that was also found in A/C intersubtype recombinants. While our analysis demonstrated the conservation of CKII domains in HIV-1 group M and O isolates, the number of potential CKII phosphorylation sites was variable in SIVcpz sequences. The results of this study will provide a basis for future mutagenesis studies to examine the role of certain amino acid residues in the structure and function of Vpu.

Phylogenetic analysis of partial env sequences of HIV-1 isolates from Cyprus and Greece suggested the existence of a distinct subtype of the virus, designated as I. We examined whether this subtype represents a distinct group, or a mosaic consisting of previously characterized subtypes. The full-length sequences under consideration were recovered from serum samples of "subtype I" obtained from two nonepidemiologically linked HIV-1-infected subjects in Greece. The first subject was an intravenous drug user (IDU), while the second was a vertically infected child born in 1984 whose parents were both IDUs. A variety of methods, such as diversity plots as well as phylogenetic and informative site analyses, were used to classify the DNA sequences. Subsequent detailed analysis revealed a unique genomic organization composed of alternating portions of subtypes A, G, and I. The two Greek isolates formed a distinct group in most of the pol, gp120, and gp41 regions, and in the vif/vpr, vpu, LTR, and 5' terminus of nef. In contrast, different parts of env and gag as well as the 3' pol region, and the first exons of tat and rev, appeared to have arisen from subtypes A and G. Our results indicate that subtype I, which was probably circulating in Greece in the early 1980s, is a triple mosaic consisting of A, G, and I sequences.

The epidemic of human immunodeficiency virus type 1 (HIV-1) in Argentina is distinctive in that many infections are caused by subtype BF recombinant viruses. To determine their demographic history, we estimated the evolutionary rate, mode of population growth, and age of genetic diversity among 40 BF vpu sequences. This revealed one of the highest substitution rates reported for HIV-1, at 10.793 × 10 −3 substitutions per site per year, and a very rapid rate of population growth, with an initial mean epidemic doubling time of 3.72 months. This rapid population growth is compatible with an elevated fitness for subtype BF compared to that for “pure” B and F viruses.

The chimeric simian-human immunodeficiency virus SHIVKU-1, bearing the envelope of human immunodeficiency virus type 1 (HIV-1), causes fulminant infection with subtotal loss of CD4+ T cells followed by development of AIDS in intravaginally inoculated macaques and thus provides a highly relevant model of sexually transmitted disease caused by HIV-1 in human beings. Previous studies using this SHIV model had shown that thevpu and nef genes were important in pathogenesis of the infection, and so we deleted portions of these genes to create two vaccines, ΔvpuΔnefSHIV-4 (vaccine 1) and ΔvpuSHIVPPc (vaccine 2). Six adult macaques were immunized subcutaneously with vaccine 1, and six were immunized orally with vaccine 2. Both viruses caused infection in all inoculated animals, but whereas vaccine 1 virus caused only a nonproductive type of infection, vaccine 2 virus replicated productively but transiently for a 6- to 10-week period. Both groups were challenged 6 to 7 months later with pathogenic SHIVKU-1 by the intravaginal route. All four unvaccinated controls developed low CD4+ T-cell counts (KU-1, and two in group 1 developed a persistent productive infection followed by development of AIDS in one. The other 10 have maintained almost complete control over virus replication even though spliced viral RNA was detected in lymph nodes. This suppression of virus replication correlated with robust antiviral cell-mediated immune responses. This is the first demonstration of protection against virulent SHIV administered by the intravaginal route. This study supports the concept that sexually transmitted HIV disease can be prevented by parenteral or oral immunization.

By animal to animal passage in rhesus and pig-tailed macaques, we developed a rhesus model of HIV-1 disease in humans. Rhesus macaques infected with a cell-free stock of SHIVKU-2 developed CD4+ T cell loss, primary lentiviral encephalitis and pneumonia, and AIDS. Six of nine rhesus macaques died within eight months post-inoculation, while the remaining three are at five, five, and eight months post-inoculation, respectively. Animals infected by either mucosal or parenteral routes of infection had a similar course of infection.

The use of Moloney murine leukemia virus (Mo-MLV)-based vectors to deliver therapeutic genes into target cells is limited by their inability to transduce nondividing cells. To test the capacity of HIV-based vectors to deliver genes into nondividing cells, we have generated replication-defective HIV type 1 (HIV-1) reporter vectors carrying neomycin phosphotransferase or mouse heat stable antigen, replacing the HIV-1 sequences encoding gp160. These vectors also harbor inactivevpr,vpu, andnefcoding regions. Pseudotyped HIV-1 particles carrying either the ecotropic or the amphotropic Mo-MLV envelope proteins or the vesicular stomatitis virus G protein were released after single or double transfections of either human 293T or monkey COS-7 cells with titers of up to 107colony-forming units per milliliter. A simple ultrafiltration procedure resulted in an additional 10- to 20-fold concentration of the pseudotyped particles. These vectors along with Mo-MLV-based vectors were used to transduce primary human skin fibroblasts and human peripheral blood CD34+cells. The HIV-1 vector system was significantly more efficient than its Mo-MLV-based counterpart in transducing human skin fibroblasts arrested at the G0/G1stage of the cell cycle by density-dependent inhibition of growth. Human CD34+cells were transduced efficiently using HIV-1 pseudotype particles without prior stimulation with cytokines.

CEM T-cells chronically infected with most HIV-1 isolates gradually cease virus production over a 4–6 week period. This is due to slow shutdown of virus replication in the majority of the cells, leading to latent infections. We identified one HIV-1 isolate (HIV213) which shut down into latency at a rate much slower than most HIV strains, requiring more than 12 weeks for the majority of the cells to become nonproductive. This indicated that genes of the virus influence the rate of shutting down, or alternatively, the length of time chronically infected cells produce virus. The viral gene(s) influencing differential rates of shutdown were mapped using chimeric viruses composed of the HIV213genome substituted with various restriction fragments from HIVMCK, which rapidly progresses into latency. We found that the 3′ region of the LTR was the major determinant influencing the rate of shutdown, but thetat/rev/vpuregion also slightly influenced this phenotype. These data show that at least these two genomic regions can influence the duration of virus production in chronically infected cells and that polymorphisms in these regions result in phenotypically divergent viruses which go into latency at different rates. It is also possible that this viral property may be an important determinant of clinical outcomes.

The human immunodeficiency virus type 1 (HIV-1) genes vpu, env, and nef have all been implicated in modulating the levels of cell surface CD4 on infected cells. To quantitatively assess the relative contribution of each gene product to the regulation of CD4 during HIV infection of Jurkat T cells and peripheral blood mononuclear cells, we have developed an infectious HIV reporter system which expresses different combinations of these genes. To distinguish infected cells in the early or late stages of infection from uninfected cells, these viruses were designed to express human placental alkaline phosphatase with the kinetics of either early or late viral genes. Flow cytometry to detect placental alkaline phosphatase and CD4 in infected cells showed that vpu, env, and nef are independently capable of down-modulation of CD4. As predicted by their respective expression patterns, nef down-modulated CD4 rapidly during the early phase of virus infection whereas vpu and env functioned late in the infection. In both Jurkat cells and peripheral blood mononuclear cells, a combination of the three genes was more efficient than any one or two genes, demonstrating that all three genes are required to achieve maximal CD4 down-modulation. In primary cells, down-modulation of CD4 was less efficient than in Jurkat cells and there was a stronger dependence on nef function for reducing cell surface CD4. HIV therefore has three genes that are able to independently down-modulate CD4; together, they can eliminate the bulk of cell surface CD4.

Chimeric primate lentiviruses composed of SIV and HIV genes may allow the analysis of the role of these discrete HIV genes in viral pathogenesis in macaque monkeys. We have constructed a chimeric virus in which the env, rev, tat, and vpu genes of HIV-1 Lai replace the env, rev, and tat genes of the SIVmac239 genome. This virus, SHIVsbg, replicates efficiently in rhesus (Indian and Chinese subspecies) and cynomolgus monkeys with viral loads in PBMC and lymph nodes of up to one infected cell per 30 cells during the acute phase of the infection. Sera from all monkeys recognize specific HIV-1 glycoproteins. The onset of lymphadenopathy in all animals was concurrent with a depletion of CD4 lymphocytes in peripheral blood. The virulence of this SHIV for rhesus and cynomolgus monkeys therefore closely parallels that of HIV-1 for human in the acute phase of the infection. Changes in the env and vpu genes of a molecular clone of HIV-1 can now be analyzed after passage in nonhuman primate species as the SHIVsbg replicates efficiently. The SHIVsbg-macaque model is an important step in the development of a readily available animal model for HIV-1 vaccine studies.

We evaluated the function of human immunodeficiency virus type 1 vpu gene in various cell lines. We established a highly sensitive system consisting of chloramphenicol acetyltransferase and reverse transcriptase assays and used it to monitor the effects of mutation of the vpu gene. In some cell lines, Vpu protein was not required at the early phase of viral replication but was important for efficient virion production. In these cells, the Vpu protein functioned effectively irrespective of the presence of intact env gene products. Likewise, CD4 gene expression had no effects on Vpu function. In the other cell lines tested, Vpu protein was not important for virion release, and the vpu mutant clone generated a normal level of progeny virions upon transfection.

The polymerase chain reaction (PCR) fails to detect HIV-1 sequences in 5% of infected individuals. To screen for false-negative PCR tests, we developed a nonisotopic PCR assay in which sequences from the beta-globin gene and from the HIV-1 vpu-env region were coamplified with biotinylated and fluorescein-labeled primers, respectively. Coamplified products were reacted with specific internal digoxigenin-labeled RNA probes. Hybrids were detected in a microtiter plate coated with streptavidin or anti-fluorescein antibody, with enzyme-labeled anti-digoxigenin antibody. After the optimization of the coamplification conditions, the assay could detect 5 proviral DNA copies in a lysate from 100,000 peripheral blood mononuclear cells. Fifty-seven samples from 55 HIV-1-seropositive patients and 25 samples from 25 seronegative individuals were evaluated. Fifty-two samples from HIV-infected individuals were positive for HIV-1 vpu-env sequences. Three of the 5 PBMC lysates falsely negative for HIV-1 sequences had reactivities for beta-globin (3-23 fu) below that of 100,000 cells (304 fu). Nonisotopic coamplification allowed for the evaluation of the quality of specimens for PCR concurrently with the detection of the presence of viral template sequences.