Initiation of genomic plus-strand RNA synthesis from DNA and RNA templatesby a viral RNA-dependent RNA polymerase

In contrast to the synthesis of minus-strand genomic and plus-strand subgen omic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-stran d RNA synthesis in vitro have not been previously reported. Therefore, litt le is known about the biochemical requirements for directing genomic plus-s trand synthesis. Using DNA templates to characterize the requirements for R NA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minu s-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Ge nomic plus-strand initiation was specific since cucumber mosaic virus minus -strand RNA templates were unable to direct efficient synthesis under the s ame conditions. In addition, mutational analysis of the minus-strand templa te revealed that the -1 nontemplated nucleotide, along with the +1 cytidyla te and +2 adenylate, is important for RNA-dependent RNA polymerase interact ion. Furthermore, genomic plus-strand RNA synthesis is affected by sequence s 5' of the initiation site.