PROJECT SUMMARY/ABSTRACT (DESCRIPTION): RNA viruses can be devastating human pathogens that impart large medical and economic burdens to society (e.g., SARS-CoV-2, influenza A virus, Ebola virus, rotavirus, etc.). While these viruses can differ quite dramatically in their pathogenesis, they share a common replication feature—they must synthesize new RNA molecules from RNA templates. Because host cell enzymes lack this activity, RNA viruses encode a specialized RNA-dependent RNA polymerase (RdRp). Viral RdRps are structurally- and functionally-conserved among diverse RNA viral families, and they directly catalyze all stages of viral transcription and genome replication. However, these enzymes rarely function alone in the context of infected cells. Instead, the viral RdRps are tightly regulated by other proteins in multi-subunit transcriptase/replicase complexes so as to maximize the type and timing of viral RNA synthesis. The overall objective of this application is to gain mechanistic insight into RdRp regulation for rotavirus—an 11-segmented, double-stranded (ds) RNA virus that causes life-threatening diarrhea in young children. The rotavirus VP1 RdRp functions only when bound beneath the icosahedral VP2 core shell layer of intact, or partially intact, particles. Engagement of VP1 by VP2 during early particle assembly activates the RdRp so that it functions as a replicase, converting packaged, single-stranded positive sense (+) RNA templates into dsRNA genome segments. Early assembly intermediates then morph into double-layered particles, wherein the VP2-bound, VP1 RdRp switches to a transcriptase activity, synthesizing +RNAs using dsRNA templates. Still, major gaps in knowledge remain about the structure of the VP1 RdRp as a replicase during dsRNA synthesis and its regulation by the VP2 core shell protein. Here, well-established in vitro biochemical and genetic techniques are combined with state-of-the-art structural approaches to close these gaps in knowledge and inform a deep understanding of rotavirus RdRp regulation. AIM 1 will elucidate VP2 core shell determinants critical for VP1 replicase activity, and AIM 2 will determine the first-ever in situ 3D atomic structures of VP1 as a replicase in both ice (using cryo-EM) and liquid (using a microfluidics system). The work outlined in this application is significant, as it is expected to reveal detailed structure-function information about the rotavirus RdRp that could be applied to rational antiviral drug design.