The dynamics of structural proteins during alphavirus entry and exit

Abstract

Alphaviruses are enveloped viruses that enter cells through endocytosis. The low pH in the endosome triggers a membrane fusion reaction, which is mediated by the virus fusion protein E1. E1 exists as a heterodimer with its chaperone E2 protein on the virus surface. Once exposed to low pH during entry, E1 dissociates from E2, inserts into the target membrane, and refolds into a more stable trimeric conformation to promote fusion. The driving force for E1 conformational change is not clear. In this study, we identified an interaction network that connects the E1 DI-DIII linker region with the E1 DI/II core trimer. Most single mutations of residues in this network showed reduced fusion efficiency. In addition, truncated E1 protein without the DI-DIII linker region failed to form a stable trimer or stably insert into liposomes at low pH. Together, our results suggested a critical role of the DI-DIII linker region during the low-pH dependent refolding of E1 protein, and a possible target for anti-viral strategies.;During virus replication, the virus capsid protein is released in the cytoplasm, and it assembles with the RNA genome into the viral nucleocapsid (NC) core. At the plasma membrane (PM), virus particles containing a NC core are released. In order to follow the movement of capsid proteins from its release to its final assembly into virus particles, we developed a system where the capsid protein is fluorescently labeled using a tetracysteine motif that does not affect its biological activity. In total, we identified three categories of capsid puncta. The first group consisted of rapidly moving internal puncta that co-localized with the E2 glycoprotein. The second group was immotile internal puncta that co-localized with Ras-GTPase-activating protein SH3 domain binding protein (G3BP). The third group was localized at the PM and contained E2 glycoprotein. This is the first time a system has been used to follow the activity of alphavirus capsid protein by live cell imaging, and it provides a platform for understanding the detailed mechanisms underlying the alphavirus budding process.