Rotavirus core shell protein sites that regulate intra-particle polymerase activity

Abstract

ABSTRACT Rotaviruses employ an intra-particle ribonucleic acid (RNA) synthesis mechanism, whereby the 11-segmented double-stranded RNA (dsRNA) genome is transcribed and replicated within the confines of a proteinaceous capsid. In this manner, the rotavirus RNA-dependent RNA polymerase (VP1) only functions while tethered to the inner core shell protein (VP2). Atomic models of VP1 inside of rotavirus double-layered particles show that it is bound beneath the VP2 layer, slightly off center from each icosahedral fivefold axis. The VP1-VP2 interaction interface exceeds 1,000 Å 2 in area and likely involves numerous amino acid side chain and backbone contacts. Here, we analyzed the existing structural data to predict residues of VP2 that could make regulatory contacts with VP1. To test which, if any, of these core shell sites alter the activity of the polymerase, we employed recombinant, mutant VP2 proteins (strain SA11) and an in vitro dsRNA synthesis assay. Our results showed that VP1 had significantly increased in vitro activity in the presence of four single-alanine VP2 mutants (K5A, T344A, E345A, and E365A) as compared with wild-type (WT) VP2, suggesting that altered core shell sites negatively regulate the polymerase. We also identified five alanine VP2 mutants (R4A/K5A/R6A, Q73A/L78A/E83A, K95A, L355A, and R332A/S333A) that supported significantly reduced in vitro VP1 activity, indicating that these core shell sites normally promote polymerase function. For these latter mutants, four of them (Q73A/L78A/E83A, K95A, L355A, and R332A/S333A) assembled into virus-like particles (VLPs) and retained their capacity to encapsidate VP1. Still, polymerases tethered within VLPs made with R332A/S333A VP2 were significantly reduced in their capacity to perform in vitro dsRNA synthesis as compared with those within WT VLPs. These results suggest that the lower VP1 activity in the presence of R332A/S333A VP2 is unlikely to be the result of defective core shell formation or reduced polymerase tethering. Instead, VP2 sites R332 and S333 may directly induce conformational changes in the polymerase, allowing it to mediate dsRNA synthesis. This study identified precise rotavirus core shell residues important for regulating intra-particle polymerase activity, thereby contributing mechanistic insights into a central step of the rotavirus lifecycle. IMPORTANCE Rotaviruses are important causes of severe gastroenteritis in young children. A characteristic feature of rotaviruses is that they copy ribonucleic acid (RNA) inside of the viral particle. In fact, the viral polymerase (VP1) only functions when it is connected to the viral inner core shell protein (VP2). Here, we employed a biochemical assay to identify which sites of VP2 are critical for regulating VP1 activity. Specifically, we engineered VP2 proteins to contain amino acid changes at structurally defined sites and assayed them for their capacity to support VP1 function in a test tube. Through this work, we were able to identify several VP2 residues that appeared to regulate the activity of the polymerase, positively and negatively. These results are important because they help explain how rotavirus synthesizes its RNA while inside of particles and they identify targets for the future rational design of drugs to prevent rotavirus disease.