Unilateral Synthesis of Reovirus Double-Stranded Ribonucleic Acid by a Cell-Free Replicase System

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RESUMO

A large-particle fraction obtained from reovirus-infected L cells contained both replicase and transcriptase activity. The in vitro replicase reaction slowed down soon after initiation, whereas the transcriptase reaction proceeded at an unabated rate. The replicase and transcriptase were both template-bound and could be separated from one another by controlled chymotryptic digestion followed by centrifugation in a CsCl gradient. The transcriptase was recovered as a sharp band (ρ = 1.43) and resembled virus core derived from mature virions. In contrast, replicase activity was distributed throughout the gradient, indicating that replicase is associated with structures of various density in CsCl. In subsequent experiments, the replicase product was found to be indistinguishable from the double-stranded ribonucleic acid (RNA) reovirus genome with respect to its buoyant density in cesium-salt gradients and denaturation-annealing characteristics. A “hybridization-competition” experiment in which the replicase product was denatured and annealed in the presence of an excess of plus-RNA indicated that the in vitro replicase reaction proceeded by means of a unilateral synthesis of minus-RNA upon a preexisting plus-RNA template, presumably of single-stranded form.

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