RNA-binding and capping activities of proteins in rotavirus open cores

Guanylyltransferases are members of the nucleotidyltransferase family and f unction in mRNA capping by transferring GMP to the phosphate end of nascent RNAs. Although numerous guanylyltransferases have been identified, studies which define the nature of the interaction between the capping enzymes of any origin and their RNA substrates have been limited. Here, we have charac terized the RNA-binding activity of VP3, a minor protein component of the c ore of rotavirions that has been proposed to function as the viral guanylyl transferase and to direct the capping of the 11 transcripts synthesized fro m the segmented double-stranded RNA (dsRNA) genome of these viruses. Gel sh ift analysis performed with disrupted (open) virion-derived cores and virus -specific RNA probes showed that VP3 has affinity for single-stranded RNA ( ssRNA) but nut for dsRNA. While the ssRNA-binding activity of VP3 was found to be sequence independent, the protein does exhibit preferential affinity for uncapped over capped RNA. Like the RNA-binding activity, RNA capping a ssays performed with open cores indicates that the guanylyltransferase acti vity of VP3 is nonspecific and is able to cap RNAs initiating with a G or a n A residue. These data establish that all three rotavirus core proteins, V P1, the RNA polymerase; VP2, the core capsid protein; and VP3, the guanylyl transferase, have affinity for RNA but that only in the case of the RNA pol ymerase is the affinity sequence specific.