Uriel Hazan

Anti-CXCR4 Monoclonal Antibodies Recognizing Overlapping Epitopes Differ Significantly in Their Ability To Inhibit Entry of Human Immunodeficiency Virus Type 1

ABSTRACT CXCR4 is one of two physiologically relevant human immunodeficiency type 1 (HIV-1) entry coreceptors. Studies of CXCR4 mutants have not clearly identified the determinants of coreceptor function and specificity. We therefore used a panel of monoclonal antibodies to further elucidate CXCR4 expression, structure, and function. Our findings show the existence of conformational subpopulations of CXCR4 that are in equilibrium on the cell surface but are not cell type specific as previously reported. HIV-1 X4 isolates can interact with multiple CXCR4 conformations in order to gain entry into target cells.

Human adipose cells express CD4, CXCR4, and CCR5 receptors: a new target cell type for the immunodeficiency virus-1?

The concept that adipocytes belong to an essential endocrine system with some characteristics of immune cells has recently emerged. The aim of this paper is to present evidence of the expression of CD4, CXCR4, and CCR5 receptors by human adipocytes and to test whether adipose cells support HIV entry. Primary human preadipocytes were cultured and differentiated in vitro. Expression of the three receptors on preadipocytes and adipocytes was demonstrated by reverse transcriptase‐polymerase chain reaction, immunocytochemical, and immunohistochemical analysis. Infection of adipose cells to HIV‐1 was then investigated. The measurement of the viral p24 antigen in preadipocyte culture medium showed an increase of p24 levels between 24 and 72 h postexposure and then a progressive decrease to reach a low level at 10–15 days. Ten days after the infection test, supernatant of preadipocytes contained infectious particles able to infect the susceptible T‐CD4 CEM cell line. The expression of viral proteins by adipocytes was confirmed using a fusion test. The presence of viral DNA was exhibited by gag‐specific polymerase chain reaction, supporting the hypothesis of HIV‐1 X4‐ and R5‐virus entry in preadipocytes. Adipose cells represent the first cell type that does not belong to the immune system expressing all specific HIV receptors and may represent new HIV‐1 target cells.

Characterization of two candidate genes, NCoA3 and IRF8, potentially involved in the control of HIV-1 latency

BackgroundThe persistence of latent HIV-1 reservoirs is the principal barrier preventing the eradication of HIV-1 infection in patients by current antiretroviral therapy. It is thus crucial to understand the molecular mechanisms involved in the establishment, maintenance and reactivation of HIV-1 latency. Since chromatin remodeling has been implicated in the transcriptional reactivation of the HIV-1 promoter, we assessed the role of the histone deacetylase inhibitor sodium butyrate (NaB) on two HIV-1 latently infected cell lines (U1 and ACH-2) gene expression.ResultsAnalysis of microarrays data led us to select two candidate genes: NCoA3 (Nuclear Receptor Coactivator 3), a nuclear receptor coactivator and IRF8 (Interferon Regulatory Factor 8), an interferon regulatory factor. NCoA3 gene expression is upregulated following NaB treatment of latently infected cells whereas IRF8 gene expression is strongly downregulated in the promonocytic cell line following NaB treatment. Their differential expressions were confirmed at the transcriptional and translational levels. Moreover, NCoA3 gene expression was also upregulated after treatment of U1 and ACH-2 cells with phorbol myristyl acetate (PMA) but not trichostatin A (TSA) and after treatment with NaB of two others HIV-1 latently infected cell lines (OM10.1 and J1.1). IRF8 gene is only expressed in U1 cells and was also downregulated after treatment with PMA or TSA. Functional analyses confirmed that NCoA3 synergizes with Tat to enhance HIV-1 promoter transcription and that IRF8 represses the IRF1-mediated activation through the HIV-1 promoter Interferon-stimulated response element (ISRE).ConclusionThese results led us to postulate that NCoA3 could be involved in the transcriptional reactivation of the HIV-1 promoter from latency and that IRF8 may contribute to the maintenance of the latent state in the promonocytic cell line. Implication of these factors in the maintenance or reactivation of the viral latency may provide potential new targets to control HIV-1 replication in latent viral reservoirs.

Determination of Essential Amino Acids Involved in the CD4-Independent Tropism of the X4 Human Immunodeficiency Virus Type 1 m7NDK Isolate: Role of Potential N Glycosylations in the C2 and V3 Regions of gp120

ABSTRACT Seven mutations in the C2, V3, and C3 regions of gp120 are implicated in the tropism of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. Site-directed mutagenesis revealed that three amino acids are essential to maintain this tropism, one in the C2 region and two in the V3 loop. Two mutations implied N glycosylation modifications.

Mutations in the env gene of human immunodeficiency virus type 1 NDK isolates and the use of African green monkey CXCR4 as a co-receptor in COS-7 cells

A previous report from this laboratory described the isolation of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. This independence of CD4 is due to seven mutations located in the C2, V3 and C3 regions of the gp120 protein. The present report describes the entry features of the m5NDK virus, which contains five of the seven m7NDK mutations, located in the V3 loop and C3 region. The entry of this virus is strictly CD4-dependent but it can fuse with African green monkey (agm) COS-7 cells bearing human CD4 (h-CD4). This fusion is directly due to the five mutations in the envgene. It has also been shown that entry of m7NDK is CD4-independent in COS-7 cells. Since the wild-type NDK and m7NDK viruses use the human CXCR4 protein as co-receptor, agm-CXCR4 was cloned and used in transfection and fusion inhibition experiments to show that this receptor can be used by the m5 and m7NDK viruses. The wild-type NDK virus, which does not enter COS-7 cells, can use agm-CXCR4, but only when the receptor is transfected into target cells. Although co-receptor nature and expression levels are still major determinants of virus entry, this is the first case where a few mutations in the env gene can overcome this restriction.

CXCR4 Is Down-Regulated in Cells Infected with the CD4-Independent X4 Human Immunodeficiency Virus Type 1 Isolate m7NDK

ABSTRACT Macrophages and T cells infected in vitro with CD4-dependent human immunodeficiency virus type 1 (HIV-1) isolates have reduced levels of CD4 protein, a phenomenon involved in retroviral interference. We have previously characterized the first CD4-independent HIV-1 X4 isolate m7NDK, which directly interacts with CXCR4 through its mutated gp120. We thus investigate CXCR4 expression in cells infected with this m7NDK CXCR4-dependent HIV-1 mutant. We present evidence of the down-regulation of CXCR4 membrane expression in CD4-positive or -negative cells chronically infected with the HIV-1 m7NDK, a phenomenon which is not observed in the CD4-dependent HIV-1 NDK parental strain. This down-regulation of CXCR4 was demonstrated by fluorescence-activated cell sorter analysis and was confirmed by the absence of CXCR4 functionality in m7NDK-infected cells, independently of the presence of CD4 protein. Furthermore, a drastic reduction of the intracellular level of CXCR4 protein was also observed. Reduced levels of CXCR4 mRNA transcripts were found in m7NDK-infected HeLa and CEM cells, reduced levels that could not be attributed to a reduced stability of CXCR4 mRNA. Down-regulation of CXCR4 on m7NDK-infected cells may thus be explained by transcriptional regulation.

Antiviral properties of two trimeric recombinant gp41 proteins

BackgroundAs it is the very first step of the HIV replication cycle, HIV entry represents an attractive target for the development of new antiviral drugs. In this context, fusion inhibitors are the third class of anti-HIV drugs to be used for treatment, in combination with nucleoside analogues and antiproteases. But the precise mechanism of HIV fusion mechanism is still unclear. Gp41 ectodomain-derived synthetic peptides represent ideal tools for clarifying this mechanism, in order to design more potent anti-HIV drugs.ResultsTwo soluble trimeric recombinant gp41 proteins, termed Rgp41B and Rgp41A were designed. Both comprise the N- and C-terminal heptad repeat regions of the ectodomain of HIV-1 gp41, connected by a 7-residue hydrophilic linker, in order to mimic the trimeric fusogenic state of the transmembrane glycoprotein. Both recombinant proteins were found to inhibit HIV-1 entry into target cells in a dose-dependent manner. Rgp41A, the most potent inhibitor, was able to inhibit both X4 and R5 isolates into HeLa cells and primary T lymphocytes. X4 viruses were found to be more susceptible than R5 isolates to inhibition by Rgp41A. In order to elucidate how the trimeric recombinant gp41 protein can interfere with HIV-1 entry into target cells, we further investigated its mode of action. Rgp41A was able to bind gp120 but did not induce gp120-gp41 dissociation. Furthermore, this inhibitor could also interfere with a late step of the fusion process, following the mixing of lipids.ConclusionTaken together, our results suggest that Rgp41A can bind to gp120 and also interfere with a late event of the fusion process. Interestingly, Rgp41A can block membrane fusion without preventing lipid mixing. Although further work will be required to fully understand its mode of action, our results already suggest that Rgp41A can interfere with multiple steps of the HIV entry process.

TRIM5α is a SUMO substrate

BackgroundThe TRIM5α restriction factor interferes with retroviral infections by inhibiting an early step of viral replication. TRIM5α activity was recently proposed to be regulated by the SUMO machinery and one SUMO consensus conjugation site as well as three putative SUMO interacting motifs (SIMs) were identified within TRIM5α sequence. Whereas mutation of the SIM sequences was found to abolish TRIM5α antiviral activity, mutation of the consensus SUMO conjugation site did not affect its restriction capacity, although this putative site has never been shown to be actually a SUMO substrate.FindingsHere we further demonstrate that TRIM5α relies on the SUMO machinery to promote restriction, since SUMO1 overexpression enhances TRIM5α-mediated retroviral inhibition whereas knockdown of SUMO1 or E2 SUMO conjugating enzyme Ubc9 prevents restriction. Furthermore, we show for the first time that TRIM5α is SUMOylated both in vitro and in cellulo and that Lysine 10 is the main SUMOylation site. Mutation of the consensus SUMO conjugation motif in position 10 abrogated SUMOylation at this position, but did not disrupt TRIM5α antiviral activity.ConclusionsAltogether, our results confirm that the SUMO machinery is involved in TRIM5α-mediated retroviral restriction, and demonstrate that TRIM5α is a SUMO 1 and SUMO 2 substrate. The inability to abrogate TRIM5α antiviral activity by mutating its main SUMO conjugation motif supports the notion that non-covalent interaction with SUMO or SUMOylated proteins rather than TRIM5α direct SUMOylation is required.

A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression

According to several studies, the HIV-1 envelope gp120 protein and the co-receptor CXCR4 play an essential role in HIV-1 induced cell toxicity. Characterisation of the CD4-independent m7NDK isolate provided the opportunity of studying the effects of direct interactions between m7NDK gp120 and CXCR4. Therefore, an inducible expression system was designed enabling synthesis of HIV-1 Env proteins upon doxycycline induction. Analysis of the expression of the env gene of the m7NDK HIV-1 isolate revealed, unexpectedly, that even long-term expression of m7NDK gp120 did not result in cytotoxycity in CXCR4-positive or -negative cell lines. This is the first report of a CD4-independent HIV-1-protein inducible expression regulated through the Tet-On system and by an alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool to undertake analysis of HIV Env protein expression.

XMRV low level of expression in human cells delays superinfection interference and allows proviral copies to accumulate

Xenotropic Murine leukemia virus-Related Virus (XMRV) directly arose from genetic recombinations between two endogenous murine retroviruses that occurred during human xenografts in laboratory mice. Studies on XMRV could thus bring clues on how a new retrovirus could circumvent barrier species. We observed that XMRV exhibits a weak promoter activity in human cells, similar to the transcription level of a Tat-defective HIV-1. Despite this low fitness, XMRV can efficiently propagate through the huge accumulation of viral copies (≈40 copies per cell) that compensates for the low expression level of individual proviruses. We further demonstrate that there is an inverse relationship between the maximum number of viral copies per infected cell and the level of viral expression, which is explained by viral envelope interference mechanisms. Low viral expression compensation by viral copy accumulation through delayed interference could a priori contribute to the propagation of others viruses following species jumps.