Graduate Program in Cell and Molecular Biology, University of Vermont, Burlington, VT 05405, USA.
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA.
Viruses. 2014 Mar 7;6(3):1078-90. doi: 10.3390/v6031078.
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.
人类免疫缺陷病毒 1 型(HIV-1)的传播主要通过称为病毒学突触的细胞间接触发生。受感染和未受感染细胞之间这些短暂附着的形成可导致病毒颗粒的传播,随后细胞分离。或者,细胞可以融合,从而形成合胞体。富含 HIV-1 病毒粒子并积极募集到病毒组装部位的小支架蛋白 tetraspanins 已被发现可负调节 HIV-1Env 诱导的细胞-细胞融合。然而,这些跨膜蛋白如何抑制膜融合目前尚不清楚。作为阐明 tetraspanins(例如 CD9 和 CD63)抑制融合机制的第一步,我们试图确定它们作用的融合过程的阶段。使用化学上位性方法,将四种融合抑制剂与 CD9 过表达串联使用。发现过表达 CD9 的细胞对针对发夹前和半融合中间体的抑制剂敏感,而对孔扩展阶段的抑制剂不敏感。与基于显微镜的染料转移测定的结果相结合,该测定揭示了 CD9 和 CD63 诱导的半融合停滞,我们的研究强烈表明 tetraspanins 在从半融合到孔打开的转变过程中阻断 HIV-1 诱导的细胞-细胞融合。