Replication of the genomic RNA of a positive-strand RNA animal virus from negative-sense transcripts.

Studies of RNA replication among the positive-strand RNA animal viruses have been hindered by the apparent inability of their RNA-dependent RNA polymerases to initiate replication on the corresponding negative-sense RNAs. However, here I report that in the case of the nodavirus flock house virus (FHV), which has a bipartite positive-sense RNA genome, the viral RNA replicase can replicate a negative-sense transcript of the genome segment that encodes the viral capsid proteins. For this work, the FHV replication cycle was experimentally reconstructed in baby hamster kidney cells that were transfected with specialized transcription plasmids designed to direct the synthesis of RNAs which corresponded closely to the two genome segments of FHV. The RNA replicase encoded by the larger genome segment could utilize either the positive or the negative strand of the smaller segment as a template, and it catalyzed RNA replication to produce similar RNA products in the two situations. Surprisingly, studies of the nucleotide sequences that were required for replication showed that the 3' end of the negative-strand RNA contained only a minimal cis-acting signal. The success of these experiments will facilitate further studies of the cis- and trans-acting factors involved in the recognition and replication of negative-sense RNA in this system.