In vitro particle-associated uridyltransferase activity of the rotavirus VP1 polymerase

Abstract

Rotaviruses are 11-segmented, double-stranded RNA (dsRNA) viruses with a unique intra-particle RNA synthesis mechanism. During genome replication, the RNA-dependent RNA polymerase (VP1) performs minus-strand RNA (-ssRNA) synthesis on positive-strand RNA (+ssRNA) templates to create dsRNA segments. Recombinant VP1 catalyzes -ssRNA synthesis using substrate NTPs in vitro, but only when the VP2 core shell protein or virus-like particles made of VP2 and VP6 (2/6-VLPs) are included in the reaction. The dsRNA product can be labeled using [α32P]-UTP and separated from the input +ssRNA template by polyacrylamide gel electrophoresis. Here, we report the generation of [α32P]-labeled rotavirus +ssRNA templates in reactions that lacked non-radiolabeled NTPs but contained catalytically-active VP1, 2/6-VLPs, and [α32P]-UTP. Non-radiolabeled UTP competed with [α32P]-UTP to decrease product levels, whereas CTP and GTP had little effect. Interesting, ATP stimulated [α32P]-labeled product production. These results suggest that rotavirus VP1 transferred [α32P]-UMP onto viral + ssRNA in vitro via a particle-associated uridyltransferase activity.