Biomedical subjects

M Benkirane

Publications and source records attributed to M Benkirane.

At least 19 recordsLinked to original sources

PubMed2001

Interaction between cyclin T1 and SCF(SKP2) targets CDK9 for ubiquitination and degradation by the proteasome.

CDK9 paired with cyclin T1 forms the human P-TEFb complex and stimulates productive transcription through phosphorylation of the RNA polymerase II C-terminal domain. Here we report that CDK9 is ubiquitinated and degraded by the proteasome whereas cyclin T1 is stable. SCF(SKP2) was recruited to CDK9/cyclin T1 via cyclin T1 in an interaction requiring its PEST domain. CDK9 ubiquitination was modulated by cyclin T1 and p45(SKP2). CDK9 accumulated in p45(SKP2-/-) cells, and its expression during the cell cycle was periodic. The transcriptional activity of CDK9/cyclin T1 on the class II major histocompatibility complex promoter could be regulated by CDK9 degradation in vivo. We propose a novel mechanism whereby recruitment of SCF(SKP2) is mediated by cyclin T1 while ubiquitination occurs exclusively on CDK9.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2000

Selective CXCR4 antagonism by Tat: implications for in vivo expansion of coreceptor use by HIV-1.

Chemokines and chemokine receptors play important roles in HIV-1 infection and tropism. CCR5 is the major macrophage-tropic coreceptor for HIV-1 whereas CXC chemokine receptor 4 (CXCR4) serves the counterpart function for T cell-tropic viruses. An outstanding biological mystery is why only R5-HIV-1 is initially detected in new seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus emerges in a minority of patients and only in the late stage of disease, suggesting that early negative selection against HIV-1-CXCR4 interaction may exist. Here, we report that the HIV-1 Tat protein, which is secreted from virus-infected cells, is a CXCR4-specific antagonist. Soluble Tat selectively inhibited the entry and replication of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs). We propose that one functional consequence of secreted Tat is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.

Antiviral Agents↗

PubMed2000

CD4+ T cell surface CCR5 density as a determining factor of virus load in persons infected with human immunodeficiency virus type 1.

The intensity of expression of the chemokine receptor CCR5 is involved in in vitro cell infectability by human immunodeficiency virus (HIV)-1 R5 isolates. Because CCR5 expression varies among individuals, the hypothesis that this expression could determine virus load in HIV-1-infected persons was tested. The mean number of CCR5 molecules per cell was measured on peripheral blood CD4+ T lymphocytes (CCR5 density) from HIV-1-infected, asymptomatic, nontreated adults. There was a strong correlation between HIV RNA plasma level and CCR5 density (P=.009) that was independent of cell activation and was not due to an HIV-induced CCR5 up-regulation. These data are compatible with the hypothesis that CCR5 density is a key factor governing cell infectability and in vivo virus production and explain the protective effect of the Delta32CCR5 deletion, which results in low CCR5 expression. CCR5 density might be of critical predictive value in HIV infection.

Acquired Immunodeficiency Syndrome↗

PubMed2000

Tat protein of human immunodeficiency virus type 1 induces interleukin-10 in human peripheral blood monocytes: implication of protein kinase C-dependent pathway.

The clinical manifestations observed in human immunodeficiency virus type 1 (HIV-1)-infected patients are primarily due to the capacity of the virus and its components to inactivate the immune system. HIV-1 Tat protein could participate in this immune system disorder. This protein is secreted by infected cells of HIV-infected patients and is free in the plasma, where it can interact and be taken up by both infected and noninfected cells. In asymptomatic patients infected by HIV-1, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with disease progression to AIDS. In the present work, we tested the capacity of Tat to induce IL-10 production by peripheral blood monocytes of healthy donors. The results show that Tat causes the production of IL-10 in a dose- and stimulation time-dependent manner. Investigations of the mechanisms involved in signal transduction show that (i) the calcium pathway is not or only slightly involved in Tat-induced IL-10 production, (ii) the protein kinase C pathway plays an essential role, and (iii) monocyte stimulation by Tat results in the intranuclear translocation of transcription factor NF-kappaB and in the induction of phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2; activation of these two potential substrates of protein kinase C is required for the production of IL-10. Finally, our results suggest that the effect of Tat is exerted at the membrane level and that the active domain is located within N-terminal residues 1 to 45. This production of IL-10 induced by Tat could participate in the progression of HIV infection to AIDS.

Calcium↗

PubMed1999

HIV-1 tat transcriptional activity is regulated by acetylation.

The human immunodeficiency virus (HIV) trans- activator protein, Tat, stimulates transcription from the viral long-terminal repeats (LTR) through an RNA hairpin element, trans-activation responsive region (TAR). We and others have shown that trans-activator protein (Tat)-associated histone acetyltransferases (TAHs), p300 and p300/CBP-associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV-1 LTR. Here, we show that p300 and PCAF also directly acetylate Tat. We defined two sites of acetylation located in different functional domains of Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV-1 LTR. Synergism was TAR-dependent and required the intact presence of both Lys28 and Lys50. Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat-associated kinase, CDK9/P-TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation. These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD-kinase and release of Tat from TAR RNA.

3T3 Cells↗

PubMed1998

Activation of integrated provirus requires histone acetyltransferase. p300 and P/CAF are coactivators for HIV-1 Tat.

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. We show here that a nuclear histone acetyltransferase activity associates with Tat. Intracellularly, we found that Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR was linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, we delineated a requirement for the HAT component from the latter but not the former in Tat function. Finally, we observed that Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR.

3T3 Cells↗

PubMed1998

Interaction of the second coding exon of Tat with human EF-1 delta delineates a mechanism for HIV-1-mediated shut-off of host mRNA translation.

HIV-1 Tat has pleiotropic functions. While its most studied role is to activate transcription from the retroviral long terminal repeat (LTR)-promoter, Tat also has functions as a secretable growth factor, a T-cell activator, and an inducer of cellular apoptosis, amongst others. For its transcriptional function, the first coding exon of Tat appears wholly sufficient; however, lentiviruses (HIVs and SIVs) maintain and conserve a second coding exon for Tat. While the function(s) of the second exon of Tat has remained largely unknown, its integrity in lentiviral genomes suggests biological importance, possibly a role in non-transcriptional activities. To understand better the biology of the second exon of Tat in HIV-1 infection of cells, we have searched for cellular proteins that bind specifically to this protein domain. Here, we report that the human translation elongation factor 1-delta (EF-1 delta) binds to the second exon of HIV-1 Tat. Interaction between Tat and EF-1 delta dramatically reduces the efficiency of the translation of cellular, but not viral, mRNAs. These findings suggest that a non-transcriptional activity of Tat modulates cellular protein synthesis, thereby affecting the metabolism of host cells.

Amino Acid Sequence↗

PubMed1997

Mechanism of transdominant inhibition of CCR5-mediated HIV-1 infection by ccr5delta32.

Human chemokine receptor 5 (CCR5) functions as a co-receptor for Human immunodeficiency virus (HIV-1) infection. CCR5 is a seven-transmembrane cell surface receptor. Recently, a naturally occurring mutation of CCR5, ccr5Delta32, has been described. A small number of Caucasians are homozygously ccr5Delta32/ccr5Delta32, while a larger number of individuals are heterozygously CCR5/ccr5Delta32. The ccr5Delta32/ccr5Delta32 genotype has been linked to a phenotype that is "highly" protected from HIV-1 infection. On the other hand, several studies have shown that the CCR5/ccr5Delta32 genotype confers "relative" protection from AIDS with onset of disease being delayed by 2-4 years. Although it is known that peripheral blood lymphocytes from heterozygous individuals (CCR5/ccr5Delta32) support ex vivo HIV-1 replication at a reduced level compared with CCR5/CCR5 cells, the molecular basis for this observation is unknown. Here we report on events that post-translationally modify CCR5. We show that CCR5 progresses through the endoplasmic reticulum prior to appearing on the cell surface. Mature CCR5 can be post-translationally modified by phosphorylation and/or co-translationally by multimerization. By contrast, mutant ccr5Delta32, although retaining the capacity for multimerization, was incapable of being phosphorylated. ccr5Delta32 heterocomplexes with CCR5, and this interaction retains CCR5 in the endoplasmic reticulum resulting in reduced cell surface expression. Thus, co-expression in cells of ccr5Delta32 with CCR5 produces a trans-inhibition by the former of ability by the latter to support HIV-1 infection. Taken together, our findings suggest CCR5/ccr5Delta32 heterodimerization as a molecular explanation for the delayed onset of AIDS in CCR5/ccr5Delta32 individuals.

Chemokine CCL4↗

PubMed1997

Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR.

TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double-stranded RNA-activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase which has anti-viral and anti-proliferative effects. In this study we show that TRBP is a cellular down-regulator of PKR function. Assaying expression from an infectious HIV-1 molecular clone, we found that PKR inhibited viral protein synthesis and that over-expression of TRBP effectively countered this inhibition. In intracellular and in cell-free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding-independent pathway. Biologically, TRBP serves a growth-promoting role; cells that overexpress TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation.

3T3 Cells↗

PubMed1996

Binding of HIV-1 virions or gp120-anti-gp120 immune complexes to HIV-1-infected quiescent peripheral blood mononuclear cells reveals latent infection.

HIV-1-infected quiescent CD4+ cells harbor the virus in an inactive state until subsequent activation. The possibility that HIV-1 itself and the virus envelope glycoprotein 120 (gp120) might be important agents of this activation was investigated. The present data indicate that binding of heat-inactivated HIV-1 (iHIV-1) to infected resting PBMCs was sufficient to activate NF-kappa B and AP-1, to induce transition from the G0/G1 stage of the cell cycle to the S/G2/M stage, to induce cell surface expression of CD25, to stimulate provirus integration, and to commit cells to produce virus. The cumulative amount of HIV-1 produced by iHIV-1-stimulated cells strictly depended on the concentration of p24gag in the virion preparations used for stimulation. Moreover, virus production was not evidenced in infected resting cells exposed to iHIV-1 previously incubated with soluble CD4 (sCD4), indicating that activation requires a contact between HIV-1 envelope glycoproteins and cell surface CD4. Although soluble gp120 did not stimulate virus production, we found that transition to the S/G2/M stage of the cell cycle, cell surface expression of activation Ags, and virus production were stimulated by cross-linking of CD4 by gp120-anti-gp120 immune complexes. Finally, incubation of gp120-anti-gp120 immune complexes with sCD4 inhibited these effects. These findings suggest that virions and gp120 anti-gp120 immune complexes found in infected patients at all times of infection can stimulate virus production in CD4+ cells harboring HIV-1 in an inducible state.

Antigen-Antibody Complex↗

PubMed1996

HIV induces activation of phosphatidylinositol 4-kinase and mitogen-activated protein kinase by interacting with T cell CD4 surface molecules.

T cell surface CD4 molecules act as co-receptors that amplify the T cell receptor (TcR)/CD3-induced signal transduction by a mechanism that requires the interaction of CD4 with p56lck tyrosine kinase (Veillette et al.; Nature 1989 338:257). Here, we demonstrate that in the absence of TcR signaling, heat-inactivated HIV-1 (HIV-HI) also elicits a cascade of events generally considered to convey a positive signal, such as protein tyrosine phosphorylation, phosphatidylinositol 4-kinase and mitogen-activated protein kinase activation. These results contribute to understand better the control that HIV may exert on its own replication or on T cell apoptosis by modulating the activation status of its target cells through its interaction with T cell surface CD4 molecules.

1-Phosphatidylinositol 4-Kinase↗

PubMed1996

Inhibition of human immunodeficiency virus type 1 production in infected peripheral blood mononuclear cells by human leukocyte antigen class I-specific antibodies: evidence for a novel antiviral mechanism.

A well-characterized mechanism by which anti-HLA class I monoclonal antibodies (MAb) inhibit human immunodeficiency virus type 1 (HIV-1) propagation in in vitro cell cultures is the neutralization of the virus through interactions with HLA molecules associated with the virion envelope. Yet, the possibility that another mechanism of inhibition might affect a postbinding stage of the virus life cycle has been strongly suggested by our previous investigations. To demonstrate that the interaction of MAb B1-1G6 with the light chain of cell surface-expressed HLA class I molecules inhibits a postbinding step of the HIV-1 life cycle, peripheral blood mononuclear cells (PBMCs) were exposed to viruses grown in HLA class I-negative, CD4-positive cells (these viruses, which did not carry HLA class I molecules, cannot be neutralized by anti-HLA MAb during the first round of infection), and PCR was used at various times postexposure to search for the different forms of HIV-1 DNA and RNA in virus-exposed PBMCs cultured in either the presence or [correction of] absence of MAb B1-1G6. Although viral DNA was found in MAb B1-1G6-treated cells, spliced HIV-1 mRNA could not be detected in those cells. In contrast, HIV-1 gene expression was found in HIV-1-infected PBMCs treated with B9-12-1, another HLA class I-specific MAb which prevents infection of cells by cell-free viruses but which fails to inhibit cell-to-cell transmission of HIV-1. These results highlight a second antiviral mechanism by which anti-HLA MAb inhibit in vitro HIV-1 propagation.

Antibodies↗

PubMed1995

Functional epitope analysis of the human CD4 molecule: antibodies that inhibit human immunodeficiency virus type 1 gene expression bind to the immunoglobulin CDR3-like region of CD4.

We recently demonstrated that monoclonal antibody (MAb) 13B8-2, specific for the immunoglobulin (Ig) complementary determining region 3 (CDR3)-like region of the CD4 molecule, inhibits viral transcription in human immunodeficiency virus (HIV)-infected CEM cells and HIV type 1 (HIV-1) promoter activity. Here, we have studied the capacity of several MAb specific for the D1 domain of CD4, including anti-CDR2-like (Leu-3a and ST4) and anti-CDR3-like (13B8-2 and ST40) MAb, and for the D2 domain of CD4 (BL4) to inhibit both provirus transcription in HIV-1LAI-infected CEM cells and transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the HIV-1 long terminal repeat in transiently transfected CEM cells. We found that HIV-1 promoter activity and provirus transcription are inhibited only by MAb that bind to the CDR3-like region in domain 1 of CD4. Moreover, we demonstrated that the Fab fragment of an anti-CDR3-like region-specific anti-CD4 MAb is a powerful inhibitor of HIV-1 promoter activity. These results have implications for understanding the role of the CDR3-like region in CD4 T-cell signaling, which controls provirus transcription.

Antibodies, Monoclonal↗

PubMed1995

The cytoplasmic tail of CD4 is required for inhibition of human immunodeficiency virus type 1 replication by antibodies that bind to the immunoglobulin CDR3-like region in domain 1 of CD4.

Monoclonal antibodies (MAb) directed against the immunoglobulin complementary determining region 3 (CDR3)-like region of the CD4 molecule inhibit human immunodeficiency virus type 1 (HIV-1) transcription. We report here data showing that the cytoplasmic tail of CD4 is required for such inhibition to be achieved. To this aim, we studied the effect of MAb 13B8-2 treatment on (i) HIV-1 production in A2.01 cells, which express different forms of the CD4 gene, (ii) Tat-induced HIV-1 promoter activation, and (iii) mitogen-activated protein kinase (MAPK) activation, which is induced in CD4-positive cells by HIV-1 cross-linking of CD4. Inhibition of HIV production by 13B8-2 MAb treatment was consistently observed in cells expressing wild-type CD4 and cells expressing a hybrid CD4-CD8 molecule (amino acids 1 to 177 of CD4 fused to the hinge, transmembrane, and cytoplasmic domains of CD8). However, no delay in HIV-1 production was observed in cells expressing a truncated CD4 which lacks the cytoplasmic domain (CD4.401). Chloramphenicol acetyltransferase assays demonstrated that Tat-dependent activation of the HIV-1 long terminal repeat promoter was inhibited by MAb 13B8-2 in A2.01/CD4 and A2.01/CD4-CD8 but not in A2.01/CD4.401 cells. Finally, we found that MAb 13B8-2 treatment inhibited the activation of MAPK induced in A2.01/CD4 and A2.01/CD4-CD8 following cross-linking of CD4 by HIV-1.

Antibodies, Monoclonal↗

PubMed1994

The cytoplasmic domain of CD4 plays a critical role during the early stages of HIV infection in T-cells.

The role played by the cytoplasmic domain of the CD4 molecule in the process of HIV infection was investigated, using A2.01 cells which express different forms of the CD4 gene. A delay in HIV production was consistently observed in cells expressing a truncated CD4 which lacks the cytoplasmic domain (CD4.401) compared with cells expressing the wild type CD4. The delay was much less in cells expressing a hybrid CD4-CD8 molecule (amino acids 1-177 of CD4 fused to the hinge, transmembrane and cytoplasmic domains of CD8). Yet the extent of viral entry and reverse transcription, monitored by semi-quantitative PCR, was similar in each cell type studied. For further study of the mechanism responsible for delayed HIV replication in the A2.01/CD4.401 cell line, cells were treated with phytohaemagglutinin (PHA), 24 h after HIV infection. Under such experimental conditions HIV production was detected at the same time in the culture supernatants of A2.01/CD4 and A2.01/CD4.401 cells. Moreover, we found that CD4 oligomerization by HIV-1 induced NF-kappa B translocation in A2.01/CD4 and A2.01/CD4-CD8 but not in A2.01/CD4.401 cells. This was consistent with CAT assay experiments which provided evidence for Tat-independent NF-kappa B mediated activation of HIV-1 LTR promoter after HIV binding to CD4 in A2.01/CD4 and A2.01/CD4-CD8 but not in A2.01/CD4.401 cells. In contrast to results published recently by Tremblay et al. (1994, EMBO J., 13, 774-783), we propose that a positive cellular signal initiated following oligomerization of the CD4 by the virus itself is involved in NF-kappa B-dependent early HIV transcription in A2.01/CD4 cells.

Base Sequence↗

PubMed1994

Involvement of human leukocyte antigen class I molecules in human immunodeficiency virus infection of CD4-positive cells.

We have studied the putative roles of human immunodeficiency virus (HIV)-associated and cell surface-expressed major histocompatibility complex class I (MHC-I) molecules in the course of the HIV life cycle by the combined use of MHC-I molecule-positive and MHC-I molecule-negative virus particles and MHC-I molecule-positive and MHC-I molecule-negative CD4+ human cells. We found (i) that several anti-MHC-I monoclonal antibodies neutralize cell infection by direct interaction with HIV-associated MHC-I antigens, (ii) that these HIV-associated MHC-I antigens are however dispensable for cell infection, and (iii) that the cell surface-expressed MHC-I molecules are unnecessary for productive infection of CD4+ human cells. These results clarify further the functions of MHC-I molecules during the HIV life cycle.

Antibodies, Monoclonal↗

PubMed1993

An antibody that binds the immunoglobulin CDR3-like region of the CD4 molecule inhibits provirus transcription in HIV-infected T cells.

We used the polymerase chain reaction (PCR) to study which step(s) of the human immunodeficiency virus type 1 (HIV-1) life cycle may be blocked following treatment of HIV-exposed CEM cells with 13B8-2, a monoclonal antibody (mAb) specific for the immunoglobulin (Ig) CDR3-like region of the CD4 molecule and able to inhibit the productive infection of CEM cells by HIV-1. The presence of viral RNA was investigated and found in 13B8-2 mAb-treated CEM cells 30 min after viral exposure; the full-length viral DNA was found at 24 h post-infection. We also found integrated forms of viral DNA at 24 h post-infection. However, the integrated provirus was transcriptionally inactive in 13B8-2 mAb-treated cells, as demonstrated by the absence of spliced HIV-1 mRNA. The lack of HIV transcription under 13B8-2 mAb treatment was confirmed by chloramphenicol acetyltransferase (CAT) assay. We conclude that the inhibition of viral gene transcription accounts for the lack of progeny virions in culture supernatants of cells treated with this anti-CD4 mAb. We also demonstrate that 13B8-2 blocks viral production from chronically infected cells and restores CD4 cell-surface expression on CEM cells containing an integrated provirus(es). We found this effect to be reversible. Moreover, we demonstrate that 13B8-2 mAb treatment is efficient on different HIV-1 and HIV-2 virus isolates. These results may have major implications for the treatment of AIDS.

Antibodies, Monoclonal↗

PubMed1993

Ig CDR3-like region of the CD4 molecule is involved in HIV-induced syncytia formation but not in viral entry.

The HIV1 envelope glycoprotein gp120 binding site has been previously mapped by genetic studies to the CDR2-like region of the first domain of the CD4 molecule. mAb reactive with epitopes linked to this region (e.g., OKT4A) inhibit both HIV entry into CD4-positive cells and syncytia formation. A second area of this domain, the CDR3-like region, has been shown to be involved in gp120-CD4 interactions, but its role remained so far unclear. We show here that a mAb specific for the CDR3-like region of the CD4 receptor, 13B8-2, actually blocks soluble gp120 binding to CD4, inhibits HIV-induced cell-cell fusion, and prevents viral production by infected cells. However, this mAb fails to inhibit the binding of viral particles to cell-surface CD4 and their entry into CD4-positive cells. These results strongly suggest i) that soluble gp120 and virion-anchored gp120 bind CD4 in distinct manners, ii) that gp120-CD4 interactions required for viral entry and syncytia formation are different, and iii) that mAb binding to the CDR3-like region of the first domain of CD4 affects a post-entry step of the HIV replicative cycle.

Antibodies, Monoclonal↗