Structural and Functional Determinants of Rotavirus NSP2 Important for Viral Factory Formation

Abstract

During their infectious lifecycle, many viruses promote the formation of intracellular viral factories, wherein they sequester the materials needed to discreetly generate new virions without detection by the host cell. For rotavirus (RV), agents of severe pediatric gastroenteritis, these structures are called viroplasms and are formed through a process known as liquid-liquid phase separation (LLPS). An essential component required for viroplasm formation by LLPS is the RV-encoded nonstructural protein 2 (NSP2), which exists as a functional octamer. The primary aim of this dissertation was to enhance our understanding of the role of NSP2 in viroplasm formation by mapping the structural and functional determinants critical for this process. Towards this goal, a recombinant virus with a single amino acid change in the extreme C-terminal region (CTR) of NSP2 was engineered (rRV-NSP2K294E) and evaluated in comparison to the wildtype control virus (rRV-WT). The rRV-NSP2K294E mutant virus was found to be severely hindered in its replication and viroplasm formation as compared to the rRV-WT control. Moreover, a recombinant protein bearing this K294E change (NSP2K294E) was diminished in its capacity for form LLPS droplets in vitro when incubated with its binding partner non-structural protein 5 (NSP5). A previous X-ray crystallography study showed that the NSP2 CTR can exhibit an open conformation, allowing it to link neighboring octamers together to form chains. We hypothesized that the NSP2 K294E change, which is located within the CTR, may have influenced inter-octamer mechanism. However, using X-ray crystallography and biochemical approaches, we did not find any evidence of inter-octamer interactions with either the wildtype protein (NSP2WT) or the NSP2K294E variant. To provide mechanistic insight into how the K294E change in NSP2 may have impacted viroplasm formation, we next performed molecular dynamics simulations of the solved NSP2K294E structure. This work revealed that the K294E change is predicted to have stabilized a rare, excited conformation of the C-terminus, which in turn could have affected its LLPS capacity. Taken together, these findings validate that the C-terminus, including the extreme CTR, is a molecular determinant of NSP2 critical for RV viroplasm formation. Such information improves the field’s understanding of the complex interactions that promote viral factory formation and could inform antiviral drug design.