Abstract
Human immunodeficiency virus type 1 (HIV-1) transmission takes place primarily through cell-cell contacts known as virological synapses. Formation of these transient adhesions between infected and uninfected cells can lead to transmission of viral particles followed by separation of the cells. Alternatively, the cells can fuse, thus forming a syncytium. Tetraspanins, small scaffolding proteins that are enriched in HIV-1 virions and actively recruited to viral assembly sites, have been found to negatively regulate HIV-1 Env-induced cell-cell fusion. How these transmembrane proteins inhibit membrane fusion, however, is currently not known. As a first step towards elucidating the mechanism of fusion repression by tetraspanins, e.g., CD9 and CD63, we sought to identify the stage of the fusion process during which they operate. Using a chemical epistasis approach, four fusion inhibitors were employed in tandem with CD9 overexpression. Cells overexpressing CD9 were found to be sensitized to inhibitors targeting the pre-hairpin and hemifusion intermediates, while they were desensitized to an inhibitor of the pore expansion stage. Together with the results of a microscopy-based dye transfer assay, which revealed CD9- and CD63-induced hemifusion arrest, our investigations strongly suggest that tetraspanins block HIV-1-induced cell-cell fusion at the transition from hemifusion to pore opening.
Highlights
Human immunodeficiency virus type 1 (HIV-1) enters target cells by fusing the viral lipid envelope with the cell membrane, allowing the release of the viral core into the cell
We and others reported that overexpression of the tetraspanins CD9 and CD63 in HIV-1-producing cells represses virus-cell and cell-cell fusion induced by Env [22,23,24]
L6, a host transmembrane tetraspanin-like protein has no such effect, despite the fact that it colocalizes with tetraspanins at the plasma membrane and is incorporated into HIV-1 particles [23,24,33]
Summary
HIV-1 enters target cells by fusing the viral lipid envelope with the cell membrane, allowing the release of the viral core into the cell (reviewed in [1]). Several ways by which HIV-1 regulates the fusogenicity of Env have been identified These include: (a) the rapid internalization of newly synthesized Env from the surface of the infected cell (reviewed in [14]); (b) an interaction between the cytoplasmic tail of the gp transmembrane domain of Env and the matrix domain of immature Gag, which strongly represses the fusogenicity of Env within the virion, and already at the virological presynapse [15,16,17,18,19]; and (c) the active recruitment of tetraspanins to viral assembly sites [20,21], where they repress cell-cell fusion [22] and, upon their acquisition by newly formed particles, virus-cell fusion [23,24]. Env-induced cell-cell fusion while applying a panel of fusion inhibitors that operate at different stages of fusion in tandem with tetraspanin overexpression (i.e., a chemical epistasis approach), as well as by using an imaging-based fluorescent dye transfer assay
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