Mechanisms of alphavirus infectivity and adaptation - Resubmission - 1

项目摘要 虫媒病毒是由昆虫传播给人类的，但控制这一过程的分子机制 病毒生命周期中的关键步骤尚未完全了解。基孔肯雅病毒（CHIKV） 几个自然适应事件，获得突变的病毒糖蛋白，增加了其 传染性和传播，然而糖蛋白如何促进体内的传染性，传播和疾病， 不清楚我们利用体内病毒进化的力量，并在β-受体中发现了一种新的进化中间体 CHIKV E1糖蛋白的c链导致蚊子中的传播增加， 复制和发病机制，表明这些糖蛋白区域是至关重要的促进病毒 传染性和疾病。本实验室的初步研究表明，E1 β-strand c在病毒粒子中起着重要作用 热稳定性、融合和胆固醇依赖性进入。此外，利用病毒进化，我们已经确定， CHIKV E1和E2之间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驱动进入昆虫和哺乳动物不同机制， 蚊子体内的温度和脂质组成驱动病毒适应。我们的总体目标是了解 CHIKV结构蛋白调节甲病毒感染性的基本机制， 在昆虫和哺乳动物中的传播，并阐明是什么驱动了糖蛋白的适应， 昆虫在这里，我们使用互补的生物化学，遗传，体外和体内方法，加上深 测序技术，以确定CHIKV结构蛋白如何起作用以促进CHIKV进入和CHIKV 发病机制和体内传播（目的1），并确定温度和宿主脂质如何影响 蚊子中的CHIKV-载体相互作用（目的2）。了解病毒如何在体内传播的 结构蛋白调节昆虫和哺乳动物的感染性，以及宿主脂质如何影响适应性， 这对了解虫媒病毒如何传播、传播和引起疾病至关重要。

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.