Structural Mechanisms of Alphavirus Membrane Fusion

甲病毒是一组包膜RNA病毒，在马和人类中引起持续性关节炎和脑炎。目前，还没有获得许可的疫苗或抗病毒疗法来预防或治愈这种疾病。包膜病毒感染的一个关键步骤是病毒和宿主细胞膜之间的膜融合，这一过程导致病毒基因组在细胞质中传递。甲病毒通过受体介导的内吞作用进入宿主细胞。病毒E1蛋白通过早期核内体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，并在出芽位点的病毒衣壳核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有力的甲病毒膜与靶脂质体融合事件。利用协调的生化、生物物理、冷冻电镜和冷冻电子断层扫描方法，我们将研究原型甲型病毒Sindbis病毒在特定融合阶段的E1和E2蛋白的分子结构。我们将根据我们的结构结果，通过生成和表征糖蛋白突变体来测试关于甲病毒膜融合机制的具体预测。我们将在重组病毒中测试关键突变的融合功能。这项研究的完成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.