Characterization of subviral particles in cells infected with simian rotavirus SA11

Abstract

Subviral particles were isolated from lysates of simian rotavirus SA11-infected cells by sedimentation through sucrose gradients and separated by equilibrium centrifugation in CsCl gradients. A cell-free system that supports rotavirus RNA replication and transcription was used to identify particles in the CsCl gradients with associated polymerase activity. These data indicated that particles with densities of 1.34 and 1.38 g/cm3 were responsible for most of the transcriptase activity present in infected cells. Electrophoretic analysis showed that particles at 1.34 g/cm3 were analogous to double-shelled virus, consisting of the inner shell proteins VP1, VP2, and VP6, the outer shell proteins VP3 and VP7, and DS RNA. Particles of 1.38 g/cm3 were similar to single-shelled virus containing the inner shell proteins and DS RNA. The pellets of the CsCl gradients were enriched for subviral particles with replicase activity. Analysis of the pellets suggested that replicase particles contain a core of VP1 and VP2 that is similar to that found in single- and double-shelled virus but contain significantly less VP6 protein per particle than those with transcriptase activity. Two particles were detected in infected cells that contain no detectable polymerase activity; one consisted primarily of the structural proteins VP2, VP3, and VP6 and the other of the nonstructural protein NS35.