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At least 19 recordsLinked to original sources
PubMed1993

Changes in the host range and growth potential of an HIV-1 clone are conferred by the vpu gene.

vpu, alone among the genes of human immunodeficiency virus type 1 (HIV-1), is unique to this virus, with no analogous reading frame evident in the genomes of either HIV-2 or the related SIVs. Effects of vpu upon levels of virus production from infected cells have been noted, but vpu is dispensable for HIV-1 replication in vitro and its significance in the natural viral life cycle is unclear. We have now identified and characterized a major influence of vpu upon the growth and host cell range of a cloned recombinant HIV-1. This effect required the presence of a second determinant mapping to the 3' end of the env gene, and was not detected in clones incorporating an env gene derived from a different strain of the virus. This indicates that important effects of vpu may have been lost in some experimental systems because of the particular viruses used, and further suggests the involvement of a determinant in the transmembrane glycoprotein of the virus in the action of vpu.

Cell Line↗
PubMed1996

High viral load and CD4 lymphopenia in rhesus and cynomolgus macaques infected by a chimeric primate lentivirus constructed using the env, rev, tat, and vpu genes from HIV-1 Lai.

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.

Animals↗
PubMed1997

Gross defects in the vpr and vpu genes of HIV type 1 cannot explain the differences in RNA copy number between long-term asymptomatics and progressors.

Disease progression in HIV-1-infected individuals is strongly associated with persistent and high numbers of HIV-1 RNA copies. We previously reported a markedly lower viral RNA load in eight long-term asymptomatics (LTAs) compared to seven matched progressors (at 1 year after seroconversion or entry in the study, p < 0.001) (Hogervorst E, et al.: J Infect Dis 1995;171:811-821). Here we extend our study to examine whether a difference in viral load can be attributed to infection by viruses having distinct vpr and vpu genes. Sequencing of vpr and vpu genes from serum samples collected at seroconversion from both long-term asymptomatics and progressors showed full-length and intact open reading frames of both genes in all subjects. At the protein level, no difference was discerned in domains of putative functional importance within Vpr and Vpu between the two groups. Phylogenetic analysis showed no clustering of LTA sequences, which interdigitated with sequences from progressors. We therefore concluded that nonprogression is not likely to be explained by deletion of vpr and vpu, or by gross sequence abnormality in these genes.

Amino Acid Sequence↗
PubMed1997

Enhancement of retroviral production from packaging cell lines expressing the human immunodeficiency type 1 VPU gene.

The HIV-1 Vpu protein stimulates virus production by enhancing the release of viral particles from infected cells. Interestingly, Vpu was also shown to enhance the release of capsids produced by gag gene contructs of other retroviruses that lack a Vpu-like activity. To investigate the effect of Vpu expression on viral particle production in retroviral packaging cell line, we developed the Damp-VpuP cell line in which vpu expression is under the control of the tetracycline-responsive promoter. Retroviral production was measured by dosage of virion-associated reverse transcriptase activity, by capsid protein immuno-detection in cell-free supernatants and by evaluating the transfer of antibiotic resistance to target cells. Induction of the Damp-VpuP cell line caused a 40-fold increase in the titer of infectious virus-like particles when compared with control cell lines. This increase in viral titer was not the result of a clonal effect nor was it a consequence of high selective pressure but rather the effect of a Vpu-mediated enhancement of viral particle production. Similar results using the third generation psi CRIP packaging cell line confirmed these findings. Constitutive expression of vpu caused a 13-fold increase in viral titer in this packaging cell line. These results indicate that the expression of HIV-1 vpu in retroviral packaging cell lines can significantly improve the titers of infectious retroviral particles.

Animals↗
PubMed1988

Identification of a protein encoded by the vpu gene of HIV-1.

Human immunodeficiency virus 1 (HIV-1) is the aetiological agent of AIDS. The virus establishes lytic, latent and non-cytopathic productive infection in cells in culture. The complexity of virus-host cell interaction is reflected in the complex organization of the viral genome. In addition to the genes that encode the virion capsid and envelope proteins and the enzymes required for proviral synthesis and integration common to all retroviruses, HIV-1 is known to encode at least four additional proteins that regulate virus replication, the tat, art, sor and 3' orf proteins, as well as a protein of unknown function from the open reading frame called R. Close examination of the nucleic acid sequences of the genomes of multiple HIV isolates raised the possibility that the virus encodes a previously undetected additional protein. Here we report that HIV-1 encodes a ninth protein and that antibodies to this protein are detected in the sera of people infected with HIV-1. This protein distinguishes HIV-1 isolates from the other human and simian immunodeficiency viruses (HIV-2 and SIV) that do not have the capacity to encode a similar protein.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Distinct effects in primary macrophages and lymphocytes of the human immunodeficiency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate.

Macrophages and lymphocytes are the two main targets for productive HIV-1 infection in vivo. To compare the effects of the "nonessential" HIV-1 accessory genes vpr, vpu, and nef on viral replication in these primary cell types, we generated a panel of mutant viruses derived from a molecularly cloned macrophage-tropic HIV-1 primary isolate. Mutant viruses had markedly different patterns of replication in macrophages, in contrast to lymphocytes in which differences were modest. Loss of vpr or vpu reduced viral antigen production in macrophages by up to 1000-fold, while replication in lymphocytes was only marginally affected. Loss of nef did not affect lymphocyte infection, but decreased replication in macrophages to a small extent. Mutation of multiple accessory genes restricted replication in both cell types, but to a much greater extent in macrophages, and frequently resulted in nonproductive infection. The degree to which replication depended on intact accessory genes varied in macrophages from different donors. The essential functions of these accessory genes in HIV-1 infection may be related to their combined effects in facilitating productive infection of macrophages.

Base Sequence↗
PubMed1992

Dual regulation of silent and productive infection in monocytes by distinct human immunodeficiency virus type 1 determinants.

The regulation of human immunodeficiency virus type 1 infection and replication in primary monocytes was investigated by mutagenesis of recombinant proviral clones containing an env determinant required for the infectivity of monocytes. Virus replication was assayed by determination of reverse transcriptase activity in culture fluids and by recovery of virus from monocytes following cocultivation with uninfected peripheral blood mononuclear cells. Three virus replication phenotypes were observed in monocytes: productive infection, silent infection, and no infection. Incorporation of the monocytetropic env determinant in a full-length clone incapable of infection or replication in primary monocytes (no infection) conferred the capacity for highly efficient virus replication in monocytes (productive infection). Clones with the env determinant but lacking either functional vpr or vpu genes generated lower replication levels in monocytes. Mutation of both vpr and vpu, however, resulted in nearly complete attenuation of virus replication in monocytes, despite subsequent virus recovery from infected monocytes by cocultivation with uninfected peripheral blood mononuclear cells (silent infection). These findings indicate a central role for the "accessory" genes vpu and vpr in productive human immunodeficiency virus type 1 replication in monocytes and indicate that vpu and vpr may be capable of functional complementation.

Amino Acid Sequence↗
PubMed1995

Function of human immunodeficiency virus type 1 Vpu protein in various cell types.

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.

3T3 Cells↗
PubMed1997

The human immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules.

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a small integral membrane phosphoprotein with two established functions: degradation of the viral coreceptor CD4 in the endoplasmic reticulum (ER) and augmentation of virus particle release from the plasma membrane of HIV-1-infected cells. We show here that Vpu is also largely responsible for the previously observed decrease in the expression of major histocompatibility complex (MHC) class I molecules on the surface of HIV-1-infected cells. Cells infected with HIV-1 isolates that fail to express Vpu, or that express genetically modified forms of Vpu that no longer induce CD4 degradation, exhibit little downregulation of MHC class I molecules. The effect of Vpu on class I biogenesis was analyzed in more detail using a Vpu-expressing recombinant vaccinia virus (VV). VV-expressed Vpu induces the rapid loss of newly synthesized endogenous or VV-expressed class I heavy chains in the ER, detectable either biochemically or by reduced cell surface expression. This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4. Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains. VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER. The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47. Based on these findings, we propose that Vpu-induced downregulation of class I molecules may be an important factor in the evolutionary selection of the HIV-1-specific vpu gene by contributing to the inability of CD8+ T cells to eradicate HIV-1 from infected individuals.

CD8-Positive T-Lymphocytes↗
PubMed1996

CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef.

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.

Alkaline Phosphatase↗
PubMed1993

Integration is essential for efficient gene expression of human immunodeficiency virus type 1.

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.

Cell Line↗
PubMed1995

Persistent infection of macaques with simian-human immunodeficiency viruses.

Chimeric simian-human immunodeficiency viruses (SHIV) containing the human immunodeficiency virus type 1 (HIV-1) tat, rev, env, and, in some cases, vpu genes were inoculated into eight cynomolgus monkeys. Viruses could be consistently recovered from the CD8-depleted peripheral blood lymphocytes of all eight animals for at least 2 months. After this time, virus isolation varied among the animals, with viruses continuing to be isolated from some animals beyond 600 days after inoculation. The level of viral RNA in plasma during acute infection and the frequency of virus isolation after the initial 2-month period were higher for the Vpu-positive viruses. All of the animals remained clinically healthy, and the absolute numbers of CD4-positive lymphocytes were stable. Antibodies capable of neutralizing HIV-1 were generated at high titers in animals exhibiting the greatest consistency of virus isolation. Strain-specific HIV-1-neutralizing antibodies were initially elicited, and then more broadly neutralizing antibodies were elicited. env sequences from two viruses isolated more than a year after infection were analyzed. In the Vpu-negative SHIV, for which virus loads were lower, a small amount of env variation, which did not correspond to that found in natural HIV-1 variants, was observed. By contrast, in the Vpu-positive virus, which was consistently isolated from the host animal, extensive variation of the envelope glycoproteins in the defined variable gp120 regions was observed. Escape from neutralization by CD4 binding site monoclonal antibodies was observed for the viruses with the latter envelope glycoproteins, and the mechanism of escape appears to involve decreased binding of the antibody to the monomeric gp120 glycoproteins. The consistency with which SHIV infection of cynomolgus monkeys is initiated and the similarities in the neutralizing antibody response to SHIV and HIV-1 support the utility of this model system for the study of HIV-1 prophylaxis.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Rapid degradation of CD4 in cells expressing human immunodeficiency virus type 1 Env and Vpu is blocked by proteasome inhibitors.

Human immunodeficiency virus (HIV) type 1 encodes three genes, Vpu, Env and Nef, that decrease cellular CD4. Vpu and Env act cooperatively to accelerate degradation of CD4 in the endoplasmic reticulum. Here we report that Vpu/Env-induced CD4 degradation is inhibited by lactacystin, a specific inhibitor of the proteasome, and by other proteasome inhibitors, but not by non-proteasome protease inhibitors. We also note that Vpu has amino acid sequence homology with a segment of IkappaB known to be involved in proteasome-mediated degradation, suggesting that HIV-1 could have transduced cellular sequences to enhance down-regulation of CD4.

Acetylcysteine↗
PubMed1992

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.

Base Sequence↗
PubMed1990

Nucleotide sequence of a Ugandan HIV-1 provirus reveals genetic diversity from other HIV-1 isolates.

A Ugandan isolate of human immunodeficiency virus type 1 (HIV-1), designated U455, was adapted to growth in U937 cells, the provirus cloned into the lambda L47.1 vector, and its DNA sequence determined. The sequences of some of the U455 genes showed a marked divergence from those of North American and other African isolates. The sequenced clone was defective with single in-phase stop codons in the vpr and env genes and frame shift, resulting in a stop codon, within the vpu gene.

Adult↗
PubMed1989

Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein.

We have performed a detailed analysis of the biochemical properties of the human immunodeficiency virus (HIV) type 1 vpu gene product to elucidate its function during virus replication. Our data suggest that vpu is posttranslationally modified by phosphorylation, since a 16-kilodalton phosphoprotein can be specifically immunoprecipitated with both a serum from an HIV-positive individual (HIV-positive serum) and a vpu-specific antiserum. In contrast, our results suggest that vpu is not glycosylated, even though the protein contains a potential glycosylation site. In vitro translation studies demonstrated that vpu is cotranslationally integrated into microsomal membranes, suggesting that vpu is an integral membrane protein. While vpu was found in significant quantities in virus-producing cells, the protein could not be detected in cell-free culture fluids and is therefore most likely not viron associated. Processing of viral precursor proteins was unaffected by the absence of vpu, and no differences were detected in the protein compositions of wild-type and mutant virions. However, virus release from cultures producing vpu-defective virus was found to be delayed, resulting in the intracellular accumulation of viral proteins. Our data suggest that vpu has a function in the release of virus particles from infected cells.

Base Sequence↗