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S Bour

Publications and source records attributed to S Bour.

36 records · Page 2Linked to original sources
PubMed1996

The human immunodeficiency virus (HIV) type 2 envelope protein is a functional complement to HIV type 1 Vpu that enhances particle release of heterologous retroviruses.

We have recently shown that the envelope glycoprotein of the ROD10 isolate of human immunodeficiency virus type 2 (HIV-2) has the ability to positively regulate HIV-2 viral particle release. The activity provided by the ROD10 Env was remarkably similar to that of the HIV-1 Vpu protein, thus raising the possibility that the two proteins act in a related fashion. We now show that the ROD10 Env can functionally replace Vpu to enhance the rate of HIV-1 particle release. When provided in trans, both Vpu and the ROD10 Env restored wild-type levels of particle release in a Vpu-deficient mutant of the NL4-3 molecular clone with indistinguishable efficiencies. This effect was independent of the presence of the HIV-1 envelope protein. The ROD10 Env also enhanced HIV-1 particle release in the context of HIV-2 chimeric viruses containing the HIV-1 gag-pol, indicating a lack of need for additional HIV-1 products in this process. In addition, we show for the first time that HIV-1 Vpu, as well as ROD10 Env, has the ability to enhance simian immunodeficiency virus (SIV) particle release. The effects of Vpu and ROD10 Env on SIV particle release were indistinguishable and were observed in the context of full-length SIVmac239 and simian-human immunodeficiency virus chimeras. These results further demonstrate that ROD10 Env can functionally complement Vpu with respect to virus release. In contrast, we found no evidence of a destabilizing activity of ROD10 Env on the CD4 molecule. HIV-1 and HIV-2 thus appear to have evolved genetically distinct but functionally similar strategies to resolve the common problem of efficient release of progeny virus from infected cells.

Animals↗
PubMed1996

The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.

Amino Acid Sequence↗
PubMed1996

The envelope glycoprotein of human immunodeficiency virus type 2 enhances viral particle release: a Vpu-like factor?

The Vpu protein is a human immunodeficiency virus type 1 (HIV-1)-specific accessory protein that is required for the efficient release of viral particles from infected cells. Even though HIV-2 does not encode Vpu, we found that this virus is nevertheless capable of efficiently releasing virus particles. In fact, the rate of virus release from HeLa cells transfected with a full-length molecular clone of HIV-2, ROD10, was comparable to that observed for the vpu+ HIV-1 NL4-3 isolate and was not further enhanced by expression of Vpu in trans. However, consistent with previous observations showing that HIV-2 particle release is Vpu responsive in the context of HIV-1/HIV-2 chimeric constructs; exchanging the gag-pol region of NL4-3 with the corresponding region from pROD10 rendered the resulting chimeric virus Vpu responsive. Our finding that the responsiveness of HIV-2 particle release to Vpu is context dependent suggested the presence of a Vpu-like factor(s) encoded by HIV-2. Using chimeric proviruses encoding HIV-2 gag and pol in the context of the HIV-1 provirus that were coexpressed with subgenomic HIV-2 constructs, we found that the HIV-2 envelope glycoprotein had the ability to enhance HIV-2 particle release with an efficiency comparable to that of the HIV-1 Vpu protein. Conversely, inactivation of the HIV-2 env gene in the original ROD10 clone resulted in a decrease in the rate of viral particle release to a level that was comparable to that of Vpu-deficient HIV-1 isolates. Providing the wild-type envelope in trans rescued the particle release defect of the ROD10 envelope mutant. Thus, unlike HIV-1, which encodes two separate proteins to regulate virus release or to mediate viral entry, the HIV-2 Env protein has evolved to perform both functions.

Brefeldin A↗
PubMed1995

The human immunodeficiency virus type 1 Vpu protein specifically binds to the cytoplasmic domain of CD4: implications for the mechanism of degradation.

We have recently demonstrated that coexpression of Vpu and CD4 in HeLa cells results in the degradation of CD4 in the endoplasmic reticulum. The sensitivity of CD4 to Vpu-mediated degradation is conferred by the presence of specific sequences located between amino acids 402 and 420 in the CD4 cytoplasmic domain. Using an in vitro translation system, we also showed that degradation of CD4 by Vpu requires the two proteins to be present in the same membrane compartment. Although these results suggest that spatial proximity between CD4 and Vpu may be critical in triggering degradation, it remains unknown whether the two molecules have the ability to interact with each other. In order to better define the mechanisms involved in CD4 degradation, we investigated the existence and functional relevance of direct interactions between CD4 and Vpu. Coimmunoprecipitation experiments showed that Vpu specifically binds to the cytoplasmic tail of CD4. This phenomenon is relevant to the mechanism of CD4 degradation since the ability of CD8/CD4 chimeric molecules and various CD4 mutants to form complexes with Vpu correlates with their sensitivity to degradation. Accordingly, we found that amino acid residues in the CD4 cytoplasmic tail previously shown to be important for degradation are necessary for Vpu binding. We further demonstrate that a deletion mutant of Vpu as well as a phosphorylation mutant, both biologically inactive with regard to CD4 degradation, retained the capacity to interact with the CD4 cytoplasmic domain. Taken together, these results indicate that Vpu binding is necessary to trigger CD4 degradation. However, the binding to target molecules is not sufficient per se to cause degradation. Interaction between CD4 and Vpu is thus likely to be an early event critical in triggering a multistep process leading to CD4 degradation.

Amino Acid Sequence↗
PubMed1995

The human immunodeficiency virus type 1 (HIV-1) CD4 receptor and its central role in promotion of HIV-1 infection.

Interactions between the viral envelope glycoprotein gp120 and the cell surface receptor CD4 are responsible for the entry of human immunodeficiency virus type 1 (HIV-1) into host cells in the vast majority of cases. HIV-1 replication is commonly followed by the disappearance or receptor downmodulation of cell surface CD4. This potentially renders cells nonsusceptible to subsequent infection by HIV-1, as well as by other viruses that use CD4 as a portal of entry. Disappearance of CD4 from the cell surface is mediated by several different viral proteins that act at various stages through the course of the viral life cycle, and it occurs in T-cell lines, peripheral blood CD4+ lymphocytes, and monocytes of both primary and cell line origin. At the cell surface, gp120 itself and in the form of antigen-antibody complexes can trigger cellular pathways leading to CD4 internalization. Intracellularly, the mechanisms leading to CD4 downmodulation by HIV-1 are multiple and complex; these include degradation of CD4 by Vpu, formation of intracellular complexes between CD4 and the envelope precursor gp160, and internalization by the Nef protein. Each of the above doubtless contributes to the ultimate depletion of cell surface CD4, although the relative contribution of each mechanism and the manner in which they interact remain to be definitively established.

Amino Acid Sequence↗
PubMed1994

HIV-1 associated down-modulation of CD4 gene expression is differentially restricted in lymphocytic and monocytic cell lines.

We previously demonstrated that chronic infection of a monocytic cell line (U-937) with human immunodeficiency virus type 1 (HIV-1) was not accompanied by down-modulation of CD4 transcription, unlike the situation with CD4+ T lymphocyte lines. To better understand the refractoriness of monocytes to alterations in levels of CD4 mRNA, we treated HIV-IIIB chronically infected U-937 cells with phorbol myristate acetate (PMA), a known stimulus of HIV gene expression. Although PMA caused a significant increase in HIV mRNA levels that was sustained over 7 days, no effect on CD4 transcript levels was noted. Clonal derivatives of HIV-IIIB-infected U-937 cells, which produced a variety of infectious and defective particles, were likewise not affected in ability to produce CD4 mRNA. To rule out the possibility that U-937 cells select out HIV-1 variants unable to modulate CD4 mRNA levels, we passaged infectious virus from a U-937 clonal derivative (UHC1) onto different monocytic and T lymphocytic cell lines. In monocytic cell lines (U-937, PLB-985, THP-1), we observed an avirulent infection that did not affect CD4 mRNA levels, whereas UHC1 infection of each of two T lymphocytic cell lines (CEM-T4, Jurkat) caused both cytopathic replication and reductions in CD4 mRNA levels. In one case (Jurkat), variants expressing low CD4 mRNA may have emerged, because the outgrowth no longer expressed viral products. In the other case (CEM-T4), high expression of viral genes was accompanied by CD4 mRNA down-modulation, suggesting either that low-CD4-expressing variants were selected that maintained viral gene expression or that CD4 gene expression was repressed by viral products.

CD4 Antigens↗
PubMed1994

Cell surface down-modulation of CD4 after infection by HIV-1.

Entry of HIV-1 into host cells is generally mediated by the cell surface CD4 receptor after specific interaction with the viral envelope glycoprotein gp120. Infection by HIV-1 commonly leads to the disappearance of CD4 from the plasma membrane, a phenomenon referred to as receptor down-modulation. This, in turn, renders cells refractory to subsequent infection by the same or other viruses that use the CD4 receptor for entry, creating a state of superinfection immunity. CD4 down-modulation is a complex process involving a variety of viral gene products, the effects of which may be manifest at different stages within the viral replication cycle. CD4 disappearance from the cell surface occurs in each of the CD4+ lymphocytes, T-cell lines, monocytic cell lines, and monocyte-derived macrophages. Internalization of CD4 can occur after binding of either gp120 alone or gp120 antigen-antibody complexes, and may also be mediated by the HIV-1 Nef gene. Other factors that cause cell surface CD4 depletion include reductions in CD4 transcript levels, impaired translation of CD4 mRNA, formation of CD4-gp160 intracellular complexes, and degradation of CD4 mediated by the HIV-1 Vpu gene.

Animals↗
PubMed1994

Correlation between high level gp160 expression and reduced CD4 biosynthesis in clonal derivatives of human immunodeficiency virus type 1-infected U-937 cells.

We have compared cytoplasmic CD4 mRNA accumulation, CD4 biosynthesis and steady-state levels of both CD4 protein and mRNA in a variety of clonal derivatives of U-937 cells, chronically infected with human immunodeficiency virus type 1 IIIB (HIV-1), that express various cellular and viral phenotypes. These phenotypes included defective processing of either gp160 or Gag-Pol, viruses with severely limited host-range, and inability to generate viral products. All clones, with the exception of the one that failed to generate viral mRNA and proteins, did not express cell surface CD4. Furthermore, each of these clones had steady-state levels of CD4 mRNA which were either equivalent to or higher than those of the parental U-937 cell line. Patterns of cytoplasmic CD4 mRNA levels resembled those of total RNA, suggesting that CD4 mRNA transport from the nucleus to the cytoplasm was unaffected by HIV-1 infection. Profiles of steady-state levels of the CD4 protein resembled those of CD4 mRNA in the UHC clones, but CD4 biosynthesis was reduced in all clones with the exception of that which failed to express viral products. This report is the first demonstration that steady-state CD4 biosynthesis is reduced in HIV-1-infected cells. In general, there was a good correlation between high levels of expression of gp160 and reduced CD4 biosynthesis. These results suggest that HIV-1 env gene products may contribute to the observed reduction in levels of CD4 biosynthesis.

Blotting, Northern↗
PubMed1993

Infection of human monocyte-derived macrophages by human immunodeficiency virus mediated by cell-to-cell transmission.

We have infected ten-day-old primary cultures of human monocyte-derived macrophages (MDM) with HIV-1 by cocultivation with chronically infected monocytic cell lines. This work has involved the U-937 monocytoid cell line, chronically infected with the HIV-IIIB strain of HIV-1 (U-937HIV IIIB) as well as a number of cell clones, termed UHC, which were derived from U-937HIV IIIB by limiting dilution. Cell-free virus, derived from each of U-937HIV IIIB cells and the UHC1 clone were noninfectious for MDM, as determined by failure to express viral p24 antigen (Ag). In contrast, viral p24 Ag production was detected in MDM that had been cocultivated with U-937HIV IIB, and with each of three UHC clones that produced infectious virus. Infection, in each case, was confirmed by polymerase chain reaction detection via the amplification of proviral DNA. In contrast, cocultivation with the UHC15.7 clone, which fails to cleave viral gp160 to its gp120 and gp41 products or the UHC8 clone, which lacks functional reverse transcriptase, did not lead to infection of MDM. Pretreatment of MDM for 2 hr with 1 microM AZT completely prevented infection by culture fluids containing HIVada, a macrophage-tropic virus, but did not affect infection mediated by cocultivation. These results suggest that cell-to-cell transmission of HIV-1, among monocyte-derived macrophages, can be mediated by proviral DNA. Moreover, gp120 at the surface of infected cells may play an important role in this process, since cell-to-cell HIV transmission could not be demonstrated with the UHC clone that is defective in cleavage of the viral envelope glycoprotein gp160.

Base Sequence↗
PubMed1993

The role and fate of the CD4 molecule in lymphocytes and monocytes infected by HIV-1.

Infection by HIV-1 of monocyte cell lines, in contrast to T lymphocytes, did not lead to decreased steady-state levels of CD4 mRNA. Similar results were also obtained using clonal derivatives of infected U-937 cells that produced either competent, highly replicative progeny viruses or defective non-infectious particles. In each case, the infected U-937 cells or clonal derivatives were found to be significantly deficient with regard to surface representation of CD4 protein, in spite of the presence of high levels of CD4 mRNA. However, both HIV-1-infected U-937 cells, as well as clonal derivatives which produced high levels of viral env mRNA and non-infectious viral structures that lacked envelope glycoproteins, contained diminished levels of OKT4-immunoprecipitable CD4 protein, in comparison with uninfected U-937 cells. Thus, expression of viral env mRNA but neither the efficient synthesis or packaging of viral glycoproteins or viral assembly is required for disappearance of cell surface CD4 to occur. Furthermore, viral gp160 co-precipitated with CD4 in both the parental and cloned cell lines. We have also shown that the generation of intracellular complexes of gp160 and CD4 is directly responsible for the disappearance of cell surface CD4 in HIV-1-infected U-937 cells. In this system, expression of gp160 was both necessary and sufficient to result in CD4 receptor down-modulation. Finally, in vitro co-translation studies revealed that the presence or synthesis of viral gp160 led to a failure to efficiently generate CD4 protein.

Blotting, Northern↗
PubMed1993

Clinical correlates and molecular basis of HIV drug resistance.

It has been widely reported that zidovudine (ZDV)-resistant variants of human immunodeficiency virus type 1 (HIV-1) can be isolated from patients undergoing prolonged therapy with this drug. At the same time, treatment of HIV-infected individuals with ZDV and other forms of nucleotide therapy, including didanosine (ddI), have enabled patients to live longer than would otherwise be the case and to enjoy improved quality of life. HIV resistance to ZDV, ddI, and other nucleosides is attributable to a series of point mutations within the pol gene of HIV-1 that encodes the viral enzyme, reverse transcriptase (RT). This is not surprising as the virus is known to replicate at high rates in infected individuals; moreover the RT that mediates transcription of proviral DNA from viral genomic RNA is known to be highly error prone. Thus, mutants of HIV-1, which possess a drug-resistance phenotype and genotype, may be expected to emerge under the selective pressure of long-term antiviral chemotherapy. This article describes a novel mutation at site 184 within the pol gene that accounts for resistance against both ddI and zalcitibine (ddC). HIV drug resistance occurs most commonly in individuals with low CD4 cell counts who have progressed to more serious forms of disease. Moreover, viruses obtained from patients with AIDS generally display higher levels of resistance, relative to pretreatment isolates, than do viruses from patients with more-limited illness. Although observations of drug resistance can be correlated with disease progression and a weakened immune system, it is still unclear whether a cause-and-effect relationship exists. Because of the error-prone nature of viral RT and the fact that the HIV-1 genome can mutate efficiently, it can be anticipated that viral drug resistance may emerge for all forms of nucleotide therapy to be offered in the future. In addition, resistance may also become apparent with regard to drugs that block HIV replication by acting at sites within the viral replication cycle other than RT.

CD4-Positive T-Lymphocytes↗
PubMed1992

Differential susceptibilities of U-937 cell clones to infection by human immunodeficiency virus type 1.

Single-cell clones derived from the U-937 monocytic cell line were studied for susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). Of four such clones, we found that three (UC12, UC14, and UC18) supported replication of HIV-1 more efficiently than parental U-937 cells, as measured by reverse transcriptase activity and p24 core antigen production. In contrast, another clone (UC11) showed only baseline infection throughout an 8-week culture period, before finally becoming positive for expression of viral antigen. This differential susceptibility to infection directly correlated with accumulation of intracellular viral DNA. Furthermore, the UC11 clone expressed lower levels of Sendai virus-inducible tumor necrosis factor alpha mRNA than did the UC12 or UC18 clones. Susceptibility to infection did not correlate with expression of cell surface CD4, since all clones expressed similar levels of CD4 mRNA and surface membrane CD4 protein. Prior exposure of both susceptible UC18 and resistant UC11 clones to Leu3a antibody completely blocked infection by HIV-1, suggesting that no other independent receptors were recognized by the virus.

Blotting, Southern↗
PubMed1991

Diminution of CD4 surface protein but not CD4 messenger RNA levels in monocytic cells infected by HIV-1.

As expected, the productive infection of several monocytic cell lines by HIV-1 led to a diminution of cell-surface CD4 antigen. However, unlike findings reported for HIV-1-infected T cells, this decrease was not accompanied by a similar reduction in levels of CD4 transcripts. OKT4 monoclonal antibodies (MAbs) to CD4 were used in immunoprecipitation experiments to show that intracellular CD4 levels were diminished in U-937 monocytic cells that had been infected by HIV-1. These MAbs also coprecipitated viral gp120, indicating that CD4-gp120 complexes are present in infected monocytes. Our results therefore demonstrate that cell-surface down-modulation of CD4 is exclusively a post-transcriptional event in HIV-1 infected monocytic cells. These data suggest that HIV-1-mediated depletion of cell-surface CD4 in monocytes does not involve transcript down-modulation as has been reported in T lymphocytes.

Antibodies, Monoclonal↗
PubMed1991

Inhibition of gp160 and CD4 maturation in U937 cells after both defective and productive infections by human immunodeficiency virus type 1.

Our results demonstrate that the formation of intracellular complexes between the envelope glycoprotein precursor gp160 of human immunodeficiency virus type 1 and CD4 is a major event, leading to the disappearance of CD4 at the cell surface of infected U937 cells. Using both productively and defectively infected clones of U937 cells, we assessed the effect of CD4-gp160 intracellular association on the maturation of both proteins. Pulse-chase labeling followed by sequential immunoprecipitation was used to analyze the processing of both free and associated CD4 and gp160, and the results showed that the trimming, proteolytic cleavage, and degradation of gp160 were completely abrogated after intracellular binding to CD4. Similarly, the maturation process which normally transforms 80% of CD4 to a partially endoglycosidase H-resistant species was also impaired subsequent to the formation of these complexes. A comparison of gp160 maturation either in free form or as a CD4 complex revealed that neither inefficient transport nor degradation of gp160 can account for the observed blockage of CD4 maturation. Moreover, this impairment was independent of gp120 and gp41, since a defective clone of human immunodeficiency virus type 1-infected cells, unable to cleave gp160, showed binding of CD4 and inhibition of CD4 transport and maturation with the same efficiency as occurred in productively infected cells. Expression of gp160 is thus necessary and sufficient to cause CD4 receptor down-modulation for both productively and defectively infected cells.

CD4 Antigens↗
PubMed1990

High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells.

Limiting-dilution techniques were employed to derive single-cell clones from U-937 cells that had been chronically infected with human immunodeficiency virus type 1. All clones thus obtained were positive for the presence of viral antigens; however, not all of the clones produced infectious progeny virus, as detected by the presence of reverse transcriptase (RT) activity in culture fluids. Six of these clones were monitored over time to determine whether their phenotype of human immunodeficiency virus type 1 expression was stable. Three clones maintained production of RT activity at a high level and showed a very high percentage of cells positive for viral p24 antigen, as determined by indirect immunofluorescence. The other three clones showed variations in either their levels of RT activity or the number of cells positive for p24, after which they stabilized. Infectious virus could be recovered from only three clones, as assessed by coculture experiments with different cell types. Two other clones were shown to produce noninfectious viruses. Molecular analyses at the DNA, RNA, and protein levels showed extensive variations between the viral isolates recovered from each clone.

Blotting, Northern↗
PubMedDate not supplied

[Antiviral strategies in the replication of human immunodeficiency virus].

The replication cycle of any virus involves a number of steps, beginning with specific attachment to a cell surface receptor leading eventually to production of progeny viruses by infected cells. In the case of the immunodeficiency virus type-1 (HIV-1), the first step involves a specific interaction between the gp120 viral envelope surface protein and specific CD4 receptor sites at the cell surface. This is followed by penetration of the virus into cells and the formation of proviral double-stranded DNA from single-stranded viral RNA, a process mediated through the action of the viral enzyme called reverse transcriptase. This, in turn, leads to the migration of proviral DNA into the nucleus of the cell and the integration of such DNA within the host cell genome. Finally both viral RNA and viral proteins are produced by the cell's genetic apparatus and new viruses are assembled at the cell surface. The fact that integration of viral DNA into host cell chromosomes occurs means that any cellular replication event will be accompanied by replication of viral DNA. Each of these steps represents a potential target for anti-viral chemotherapy. To date, most efforts to treat HIV-associated disease have focused on the reverse transcription step. In this respect, zidovudine (AZT) has been the most widely used anti-viral drug studied. However, the relative toxicity and lack of efficiency of this drug means that our efforts to develop new therapeutic strategies to combat HIV infection must continue.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗