Structure-based studies of RNA virus entry and assembly in the native context

Electron cryomicroscopy (cryo-EM) has rapidly become the method of choice for structural studies of macromolecular complexes in vitro, but often comes at the cost of losing valuable biological context related to structure/function in the native environment of the cell. Alphaviruses, a genus of positive-strand RNA enveloped viruses with significant human health burden, are generally variable in size and shape, and thus pose a challenge to traditional cryo-EM analysis. Further, structures important to alphavirus assembly/budding and replication processes are intricately linked to the context of infected cell membranes. While the released alphavirus virion architecture has been well characterized, the structural assembly mechanism of its two icosahedral protein layers remains undefined. In chapter 2 of this thesis, we analyze the structures of purified alphavirus particles using cryo-EM imaging and single particle analysis. We discovered a subpopulation of T=3 icosahedral particles, differing from the T=4 structure of all previous alphavirus structures. We then determined near-atomic resolution structures of Mayaro and Chikungunya virus, two circulating alphaviruses in the Americas, by addressing deviations from icosahedral symmetry within each particle. In chapter 3, we studied the assembly mechanism of alphavirus particles in situ using electron cryotomography (cryo-ET) of virus-infected cells. From snapshots of virus assembly/budding events, we determined 3D structures of assembly intermediates that revealed the mechanistic roles of glycoprotein spikes and nucleocapsids in forming two-layered icosahedral particles. Further, data of CHIKV-infected cells treated with budding-inhibiting antibodies shows that spacing spikes apart to prevent their lateral interactions prevents the plasma membrane bending around NC cores, thus blocking virus budding. These findings provide the molecular mechanisms of icosahedral enveloped virus assembly/budding and budding-blocking antibodies. Finally, we present an initial in situ structure of the functional CHIKV replication complex in virus-infected cells, with a preliminary model of viral non-structural protein organization in the cell. We suggest further analysis of this replication system will reveal the functional stages of positive-strand RNA processing in the cell. Lastly, in chapter 4, we switched focus to study the near-native structures of coronavirus spike (S) trimers directly on the surface of human coronavirus (HuCoV) virions in light of the Covid-19 pandemic. S trimers protruding from the viral envelope mediate coronavirus entry and are the main target for vaccine development. We first determined the 3.4Å resolution structure of the alphacoronavirus HuCoV-NL63 prefusion S crown directly on virions without the need for recombinant, soluble protein with stabilizing mutations. Using further cryo-ET and subvolume analysis, we determined structural conformations of full-length S trimers, revealing incredible orientational flexibility mediated by a novel glycan hinge in the membrane-anchored stalk domain. Our results suggest a straightforward path for future studies of coronaviruses with external reagents such as receptors, drugs and antibodies, all in the context of pathogenicity relevant to human health

Advisor: Chiu Wah
Arvin Ann M.
Carette Jan
Sarnow P. (Peter)