TEMPLATE-DEPENDENT, IN-VITRO REPLICATION OF ROTAVIRUS RNA

A template-dependent, in vitro rotavirus RNA replication system was es tablished. The system initiated and synthesized full-length double-str anded RNAs on rotavirus positive-sense template RNAs. Native rotavirus mRNAs or in vitro transcripts, with bona fide 3' and 5' termini, deri ved from rotavirus cDNAs functioned as templates. Replicase activity w as associated with a subviral particle containing VP1, VP2, and VP3 an d was derived from native virions or baculovirus coexpression of rotav irus genes. A cis-acting signal involved in replication was localized within the 26 3'-terminal nucleotides of a reporter template RNA. Vari ous biochemical and biophysical parameters affecting the efficiency of replication were examined to optimize the replication system. A repli cation system capable of in vitro initiation has not been previously d escribed for Reoviridae.