Reovirus-Specific Enzyme(s) Associated with Subviral Particles Responds In Vitro to Polyribocytidylate to Yield Double-Stranded Polyribocytidylate·Polyriboguanylate

Abstract

In reovirus-infected cells, virus-specific particles accumulate that have associated with them a polyribocytidylate [poly(C)]-dependent polymerase. This enzyme copies in vitro poly(C) to yield the double-stranded poly(C)·polyriboguanylate [poly(G)]. The particles with poly(C)-dependent polymerase were heterogeneous in size, with most sedimenting from 300 S to 550 S . Exponential increase in these particles began at 23 h, and maximal amounts were present by 31 h, the time of onset of exponential growth of virus at 30°C. Maximal amounts of particles with active transcriptase and replicase were present at 15 and 18 h after infection. Thereafter, there was a marked decrease in particles with active transcriptase and replicase until base line levels were reached at 31 h. Thus, the increase in poly(C)-responding particles occurred coincident with the decrease in particles with active transcriptase and replicase. The requirement for poly(C) as template was specific because no RNA was synthesized in vitro in response to any other homopolymer, including 2′- O -methyl-poly(C). Synthesis was optimal in the presence of Mn 2+ as the divalent cation, and no primer was necessary for synthesis. In contrast, the dinucleotide GpG markedly stimulated synthesis in the presence of 8 mM Mg 2+ . The size of the poly(C)·poly(G) synthesized in vitro was dependent on the size of the poly(C) used as template. This suggested that the whole template was copied into a complementary strand of similar size. The T m of the product was between 100 and 130°C. Hydrolysis of the product labeled in [ 32 P]GMP with alkali or RNase T2 yielded GMP as the only labeled mononucleotide. This does indicate that the synthesis of the poly(G) strand in vitro did not proceed by end addition to the poly(C) template, but proceeded on a separate strand.