Roy Nathan H, Lambelé Marie, Chan Jany, Symeonides Menelaos, Thali Markus
Graduate Program in Cell and Molecular Biology, University of Vermont, Burlington, Vermont Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont.
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont.
J Virol. 2014 Jul;88(13):7645-58. doi: 10.1128/JVI.00550-14. Epub 2014 Apr 23.
During cell-to-cell transmission of HIV-1, viral and cellular proteins transiently accumulate at the contact zone between infected (producer) and uninfected (target) cells, forming the virological synapse. Rearrangements of the cytoskeleton in producer and target cells are required for proper targeting of viral and cellular components during synapse formation, yet little is known about how these processes are regulated, particularly within the producer cell. Since ezrin-radixin-moesin (ERM) proteins connect F-actin with integral and peripheral membrane proteins, are incorporated into virions, and interact with cellular components of the virological presynapse, we hypothesized that they play roles during the late stage of HIV-1 replication. Here we document that phosphorylated (i.e., active) ezrin specifically accumulates at the HIV-1 presynapse in T cell lines and primary CD4(+) lymphocytes. To investigate whether ezrin supports virus transmission, we sought to ablate ezrin expression in producer cells. While cells did not tolerate a complete knockdown of ezrin, even a modest reduction of ezrin expression (~50%) in HIV-1-producing cells led to the release of particles with impaired infectivity. Further, when cocultured with uninfected target cells, ezrin-knockdown producer cells displayed reduced accumulation of the tetraspanin CD81 at the synapse and fused more readily with target cells, thus forming syncytia. Such an outcome likely is not optimal for virus dissemination, as evidenced by the fact that, in vivo, only relatively few infected cells form syncytia. Thus, ezrin likely helps secure efficient virus spread not only by enhancing virion infectivity but also by preventing excessive membrane fusion at the virological synapse.
While viruses, in principal, can propagate through successions of syncytia, HIV-1-infected cells in the majority of cases do not fuse with potential target cells during viral transmission. This mode of spread is coresponsible for key features of HIV-1 pathogenesis, including killing of bystander cells and establishment of latently infected T lymphocytes. Here we identify the ERM protein family member ezrin as a cellular factor that contributes to the inhibition of cell-cell fusion and thus to suppressing excessive syncytium formation. Our analyses further suggest that ezrin, which connects integral membrane proteins with actin, functions in concert with CD81, a member of the tetraspanin family of proteins. Additional evidence, documented here and elsewhere, suggests that ezrin and CD81 cooperate to prevent cytoskeleton rearrangements that need to take place during the fusion of cellular membranes.
在HIV-1的细胞间传播过程中，病毒蛋白和细胞蛋白会在受感染（产生病毒的）细胞与未受感染（靶）细胞之间的接触区域短暂积累，形成病毒突触。在突触形成过程中，产生病毒的细胞和靶细胞中的细胞骨架重排对于病毒和细胞成分的正确靶向是必需的，但对于这些过程如何被调控，尤其是在产生病毒的细胞内，我们知之甚少。由于埃兹蛋白-根蛋白-膜突蛋白（ERM）将F-肌动蛋白与整合膜蛋白和外周膜蛋白连接起来，被整合到病毒粒子中，并与病毒前突触的细胞成分相互作用，我们推测它们在HIV-1复制后期发挥作用。在此我们证明，磷酸化（即活性）埃兹蛋白特异性地在T细胞系和原代CD4(+)淋巴细胞的HIV-1前突触处积累。为了研究埃兹蛋白是否支持病毒传播，我们试图在产生病毒的细胞中消除埃兹蛋白的表达。虽然细胞不能耐受埃兹蛋白的完全敲低，但即使在产生HIV-1的细胞中埃兹蛋白表达适度降低（约50%）也会导致释放出感染性受损的病毒颗粒。此外，当与未受感染的靶细胞共培养时，敲低埃兹蛋白的产生病毒的细胞在突触处四跨膜蛋白CD81的积累减少，并且更容易与靶细胞融合，从而形成多核巨细胞。这样的结果可能对病毒传播并非最佳，因为在体内只有相对较少的受感染细胞形成多核巨细胞这一事实证明了这一点。因此，埃兹蛋白可能不仅通过增强病毒粒子的感染性，还通过防止病毒突触处过度的膜融合来确保有效的病毒传播。
虽然原则上病毒可以通过多核巨细胞的连续形成进行传播，但在大多数情况下，HIV-1感染的细胞在病毒传播过程中不会与潜在的靶细胞融合。这种传播方式共同导致了HIV-1发病机制的关键特征，包括旁观者细胞的杀伤和潜伏感染的T淋巴细胞的建立。在此我们确定ERM蛋白家族成员埃兹蛋白是一种细胞因子，它有助于抑制细胞间融合，从而抑制过度的多核巨细胞形成。我们的分析进一步表明，将整合膜蛋白与肌动蛋白连接起来的埃兹蛋白与四跨膜蛋白家族成员CD81协同发挥作用。此处及其他地方记录的其他证据表明，埃兹蛋白和CD81共同作用以防止细胞膜融合过程中需要发生的细胞骨架重排。
