Mechanisms of alphavirus infectivity and adaptation - Resubmission - 1

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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.