Rotavirus RNA Replication: Single-stranded RNA Extends from the Replicase Particle

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Abstract
The rotavirus genome consists of 11 segments of dsRNA that are replicated asymmetrically with plus strand RNA serving as the template for minus strand RNA synthesis. In this study, we have used non-denaturing gel electrophoresis to examine subviral particles that synthesize dsRNA (replicase particles), for possible changes in structure during RNA replication. Analysis of SVPs purified from simian rotavirus SA11-infected MA104 cells and resolved on 0.6% agarose gels containing 50 mm-Tris-glycine pH 8.8 showed that the overall size of particles able to synthesize dsRNA in a cell-free system was 100 nm or more. Electrophoretic analysis of the size of replicase particles as a function of length of incubation in the cell-free system demonstrated that replicase particles decreased in size with increasing length of incubation. However, after 60 to 90 min of incubation, replicase particles no longer changed in size but were similar in size to the rotavirus single-shelled (75 nm), core (60 nm) and precore (45 nm) replicative intermediates which have been described previously. As the size of replicase particles decreased with increasing length of incubation, the number of newly made genome-length dsRNAs in the particles increased. Analysis of the RNA products detected in replicase particles showed that RNA replication is regulated such that the synthesis of full-length dsRNAs in the replicase particle proceeds from the smallest to the largest genome segments. Treatment of replicase particles with single-strand-specific RNase reduced their size to that of replicative intermediates and interfered with their ability to synthesize dsRNA, thus indicating that the plus strand RNA template for replication extends from the replicase particle. This study showed that replicase particles undergo a continuous change in size during RNA replication due apparently to plus strand RNA templates moving into the replicase particle during the synthesis of dsRNA.