Structural Mechanisms of Alphavirus Membrane Fusion

甲病毒是一组有包膜的RNA病毒，在马和人类中引起持续性关节炎和脑炎。目前，没有许可的疫苗或抗病毒治疗可用于预防或治愈该疾病。包膜病毒感染的关键步骤是病毒和宿主细胞膜之间的膜融合，这是导致病毒基因组的细胞质递送的过程。甲病毒通过受体介导的内吞作用进入宿主细胞。病毒E1蛋白通过在早期内体中酸化后的构象变化介导膜融合。另一种病毒包膜蛋白E2在病毒组装过程中作为E1蛋白的分子伴侣发挥作用，并在病毒衣壳核心在出芽位点的摄取和膜融合后的释放中发挥作用。我们已经获得了令人兴奋的，前所未有的图像，通过冷冻电子显微镜捕获了强大的甲病毒膜融合事件与靶脂质体。使用协调的生物化学，生物物理，冷冻电子显微镜，冷冻电子断层扫描方法，我们将调查的E1和E2蛋白质的原型甲病毒，辛德毕斯病毒，在特定的融合阶段的分子结构。我们将通过产生和表征糖蛋白的突变体，基于我们的结构结果测试关于甲病毒膜融合机制的具体预测。我们将在重组病毒的背景下测试关键突变的融合功能。完成这项研究将促进我们对甲病毒糖蛋白如何促进膜融合的理解。

Alphaviruses, a group of enveloped RNA viruses, cause persistent arthritis and encephalitis among horses and humans. Currently, no licensed vaccine or antiviral therapy is available to prevent or cure the disease. A critical step in enveloped virus infection is membrane fusion between the viral and the host cellular membranes, a process leading to cytoplasmic delivery of the viral genome. Alphaviruses enter the host cell via receptor- mediated endocytosis. The viral E1 protein mediates membrane fusion through conformational changes upon acidification in the early endosomes. The other viral envelope protein, E2, functions as a molecular chaperon for the E1 protein during virus assembly and plays a role in virus capsid core uptake at the budding site and release after membrane fusion. We have obtained exciting, unprecedented images that captured robust alphavirus membrane fusion events with target liposomes by cryo-electron microscopy. Using coordinated biochemical, biophysical, cryo-electron microscopy, and cryo-electron tomography methods, we will investigate the molecular structures of the E1 and E2 proteins in a prototype alphavirus, Sindbis virus, at specific fusion stages. We will test specific predictions about the alphavirus membrane fusion mechanism based on our structural results by generating and characterizing mutants of the glycoproteins. We will test the fusion function of key mutations in the context of recombinant viruses. Completing this research will advance our understanding about how alphavirus glycoproteins promote membrane fusion.