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.