Article

ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation

Groupe Virus et Immunité, Institut Pasteur, CNRS URA1930, France.
The EMBO Journal (Impact Factor: 10.75). 02/2007; 26(2):516-26. DOI: 10.1038/sj.emboj.7601509
Source: PubMed

ABSTRACT HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell-cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP-70, a key kinase regulating T-cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP-70, or expressing a kinase-dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP-70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP-70, intracellular Gag localization was impaired. ZAP-70 was required in infected donor cells for efficient cell-to-cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T-cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell-to-cell contacts.

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Available from: Andrés Alcover, Dec 23, 2013
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    • "Interestingly, Zap-70 has also been implicated in HIV-1 replication and viral spread [58,59], suggesting that DQBS may interfere with HIV-1 replication by blocking Nef-dependent Zap-70 activation in CEM-T4 cells and other T-cell hosts. Future work will address whether DQBS can similarly inhibit HIV-1 replication in macrophages, which express the Zap-70 homolog, Syk. "
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    ABSTRACT: HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.
    Retrovirology 11/2013; 10(1):135. DOI:10.1186/1742-4690-10-135 · 4.77 Impact Factor
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    • "After 1 h at 37°C, cells were fixed 10 min with 4% PFA. Cells were stained with a rabbit polyclonal anti-Gag proteins (a kind gift of Pierre Boulanger) [84] and analyzed by confocal microscopy on a Zeiss LSM700 microscope. HeLa cells were plated on glass coverslips and the day after transfected with proviral DNA coding for the pNL4-3 or the nef-deleted counterpart expressing the green fluorescent protein (GFP) in frame with the HIV p17 protein [45]. "
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    ABSTRACT: Newly synthesized HIV-1 particles assemble at the plasma membrane of infected cells, before being released as free virions or being transferred through direct cell-to-cell contacts to neighboring cells. Localization of HIV-1 Gag precursor at the cell membrane is necessary and sufficient to trigger viral assembly, whereas the GagPol precursor is additionally required to generate a fully matured virion. HIV-1 Nef is an accessory protein that optimizes viral replication through partly defined mechanisms. Whether Nef modulates Gag and/or GagPol localization and assembly at the membrane and facilitates viral cell-to-cell transfer has not been extensively characterized so far. We report that Nef increases the total amount of Gag proteins present in infected cells, and promotes Gag localization at the cell membrane. Moreover, the processing of p55 into p24 is improved in the presence of Nef. We also examined the effect of Nef during HIV-1 cell-to-cell transfer. We show that without Nef, viral transfer through direct contacts between infected cells and target cells is impaired. With a nef-deleted virus, the number of HIV-1 positive target cells after a short 2h co-culture is reduced, and viral material transferred to uninfected cells is less matured. At later time points, this defect is associated with a reduction in the productive infection of new target cells. Our results highlight a previously unappreciated role of Nef during the viral replication cycle. Nef promotes HIV-1 Gag membrane localization and processing, and facilitates viral cell-to-cell transfer.
    Retrovirology 07/2013; 10(1):80. DOI:10.1186/1742-4690-10-80 · 4.77 Impact Factor
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    • "The actin cytoskeleton plays an important role in the formation of the virological synapse [23], [27], [60], [61]. To confirm the importance of actin during cell-cell transmission under our experimental conditions we tested the effects of the actin inhibitors Lat-A and Jas on HIV cell-to-cell transmission. "
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    ABSTRACT: Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.
    PLoS ONE 01/2013; 8(1):e53138. DOI:10.1371/journal.pone.0053138 · 3.23 Impact Factor
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