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.

Small Reovirus Particles Composed Solely of Sigma NS with Specificity for Binding Different Nucleic Acids

“…The harvest from passage 2 of the Dearing strain of reovirus (type 3, cloned [5,8]) was used as stock virus in all experiments described here. The procedures for infection and incubation of cells at 300C in the presence of actinomycin D (0.15 ,ug/ml) were as described before (5)(6)(7). For labeling of reovirus proteins, Eagle Spinner medium containing 33% of the normal complement of amino acids supplemented with 7% fetal bovine serum was used.…”

“…After 12 to 13 h at 30 to 31°C, [3S]methionine at 3.8 ,uCi/ml was added. The labeled precursor was present until harvest at 23 h, when cytoplasmic extracts were prepared (6,7).…”

Small reovirus-specific particle with polycytidylate-dependent RNA polymerase activity

“…Purification of small particles with poly(C)-dependent RNA polymerase activity. We previously found that particles with poly(C)-dependent RNA polymerase activity in cytoplasmic extracts of reovirus-infected L929 mouse fibroblasts (8) or BHK-21 cells sedimented through a 40% glycerol cushion and collected in the pellet, namely in region 4 (R-4) of Fig. 1, after centrifugation for 3 h at 39,000 rpm.…”

“…The harvest from passage 2 of the Dearing strain of reovirus (type 3, cloned [7,9]) was used as stock virus in all experiments described in this report. The procedures for infection and incubation of cells at 30°C in the presence of actinomycin D (0.15 fig/ml), for growth of labeled virus and plaque assay, and for purification of cell-associated virus were as described previously (7)(8)(9)12 thionine at 2 to 13 MCi/mil was added. The labeled precursor was present until harvest.…”

“…Assay for poly(C)-dependent RNA polymerase. The assay mixture for poly(C)-dependent RNA polymerase has been described previously (8), but will be repeated because of its importance to the present report. The reaction mixture contained the experimental sample in a final volume of 0.80 ml with 80 Mmol of Tris-hydrochloride, pH 8.1, 1.9,mol of manganese chloride, 5.5 ,umol of 2-mercaptoethanol, 10 ,tg of poly(C), 0.005 ,umol of GTP, and 3 to 5 MCi of [a-32P]GTP.…”