Joyeeta Chakraborty, Baylor College of Medicine

Joyeeta Chakraborty1, Soni Kaundal1, Neetu Neetu1, Jeanette Criglar2, Sue E Crawford2, Mary K Estes2, and B.V.Venkataram Prasad1.2 1The Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, 2Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.

Virus Replication: Entry, Exit and Everything in Between

Rotavirus (RV) is a significant cause of severe gastroenteritis in children and young animals. Its replication cycle occurs in the cytoplasm, driven by specialized compartments called viroplasms, formed through liquid-liquid phase separation mediated by NSP2 and NSP5. NSP2, a highly conserved nonstructural protein, is crucial for RV replication and genome packaging. Previous studies have shown that NSP2 is an octamer, binds single-stranded RNA (ssRNA), destabilizes RNA helices to help in genome assortment, possesses nucleoside triphosphatase (NTPase) and RNA 5′ triphosphatase (RTPase) activities. NSP2 exists in two forms during RV infection, a cytoplasmic dispersed form that converts into the viroplasmic form regulated by a phosphorylation cascade. Our study focuses on elucidating the structure-based mechanisms by which NSP2 performs dual functions: first, in trafficking RV proteins and capped mRNAs from the cytoplasm into the viroplasm, and second, as a scaffolding protein in regulating the formation of replication intermediates and core particles. A key finding from our study is that we can obtain NSP2 in its monomeric form (mNSP2) in vitro. We found that mNSP2 retains NTPase and RTPase activities and exhibits autokinase activity, though its phosphorylation sites differ from those of octameric NSP2. Notably, mNSP2 assembles into an octamer in the presence of RNA and exhibits RNA chaperone activity. These observations raise an interesting question of whether this is the conformation of dispersed NSP2. Ongoing studies of mNSP2 will provide further insights into its self-assembly process and its role in RV replication. Supported by NIAID grant AI36040 (BVVP).