Mechanisms of alphavirus infectivity and adaptation - Resubmission - 1

项目总结 虫媒病毒是从昆虫媒介传播到人类的，但控制这一传播的分子机制 病毒生命周期中的关键步骤尚不完全清楚。基孔肯雅病毒(CHIKV)经历了 一些自然适应事件，获得了病毒糖蛋白的突变，这些突变增加了它的 感染性和传播性，然而糖蛋白如何在体内促进传染性、传播和疾病 不清楚。我们利用了体内病毒进化的力量，并在β中发现了一种新的进化中间体- CHIKV e1糖蛋白的c链导致在蚊子中的传播增加，并增加了 在小鼠中的复制和致病机制，表明这些糖蛋白区域对于促进病毒至关重要 传染性和疾病。我们实验室的初步研究表明，e1β-strand c在病毒粒子中起着重要的作用。 热稳定性、融合性和胆固醇依赖型进入。此外，利用我们已经确定的病毒进化 新城疫病毒E_1和E_2的宿主特异性遗传相互作用。根据这些初步研究，我们 假设CHIKV E1和E2驱动不同的进入昆虫和哺乳动物的机制，并且 蚊子体内的温度和脂肪成分推动了病毒的适应。我们的总体目标是了解 CHIKV结构蛋白调节甲型病毒感染性和免疫调节的基本机制 在昆虫和哺乳动物中的传播，并阐明是什么驱动糖蛋白在 昆虫。在这里，我们使用互补的生化、遗传、体外和体内方法，并结合深度 用测序技术确定CHIKV结构蛋白如何促进进入和CHIKV 发病机制和体内传播(目标1)，并建立温度和宿主脂类如何影响 蚊子中CHIKV与病媒的相互作用(目标2)。了解病毒的离散机制如何 结构蛋白调节昆虫和哺乳动物的感染性，以及宿主脂类如何影响适应 对于了解虫媒病毒是如何传播、传播和致病至关重要的。

PROJECT SUMMARY Arboviruses are transmitted from insect vectors to humans, yet the molecular mechanisms that govern this critical step in the viral life cycle are not completely understood. Chikungunya virus (CHIKV) has undergone several natural adaptation events, acquiring mutations in the viral glycoproteins that have increased its infectivity and transmission, yet how the glycoproteins promote infectivity, spread, and disease in vivo is unclear. We harnessed the power of in vivo viral evolution and identified a new evolutionary intermediate in β- strand c of the CHIKV E1 glycoprotein that led to increased transmission in mosquitoes and increased replication and pathogenesis in mice, indicating that these glycoprotein regions are crucial for promoting viral infectivity and disease. Preliminary studies from our lab show that E1 β-strand c plays essential roles in virion thermostability, fusion, and cholesterol-dependent entry. Moreover, using viral evolution we have identified host-specific genetic interactions between CHIKV E1 and E2. Based on these preliminary studies, we hypothesize that CHIKV E1 and E2 drive distinct mechanisms of entry into insects and mammals and that temperature and lipid composition in mosquitoes drives viral adaptation. Our overall goal is to understand the fundamental mechanisms by which the CHIKV structural proteins regulate alphavirus infectivity and dissemination in insects and mammals, and to elucidate what drives the adaptation of the glycoprotein in insects. Here, we use complementary biochemical, genetic, in vitro and in vivo approaches coupled with deep sequencing technologies to establish how the CHIKV structural proteins function to promote entry and CHIKV pathogenesis and transmission in vivo (Aim 1), and to establish how temperature and host lipids impact CHIKV-vector interactions in mosquitoes (Aim 2). Understanding the discrete mechanisms of how the viral structural proteins regulate infectivity in insects and mammals, and how host lipids impact adaptation is essential to understanding how arboviruses are transmitted, spread, and cause disease.