Synthesis of simian rotavirus SA11 double-stranded RNA in a cell-free system

Abstract

A cell-free system was developed to study the replication of simian rotavirus SA11. The components of the system included (i) subviral particles prepared from infected cells to template the synthesis of viral RNA and (ii) an mRNA-dependent rabbit reticulocyte lysate to support protein synthesis. Based upon nuclease-sensitivity, approximately 20% of the RNA made in vitro was double-stranded (dsRNA) and 80% single-stranded (ssRNA). Electrophoretic analysis of the RNA products on polyacrylamide and low pH agarose gels showed that the system supported the synthesis of 11 dsRNAs and 11 positive-sense ssRNAs that corresponded in size to authentic viral RNAs. The synthesis of dsRNA in vitro was determined to be an asymmetrical process in which a nuclease-sensitive positive-strand RNA acted as a template for the synthesis of negative-strand RNA. The system also supported the initiation of negative-strand RNA using exogenous viral positive-strand RNA as a template. Finally, analysis of subviral particles recovered from reactions suggested that viral proteins made in vitro assembled into nucleoprotein complexes which were similar to those present in infected cells. Together, these results indicate that the cell-free system supported rotavirus RNA replication, transcription and the assembly of subviral particles.