Initiation of genomic plus-strand RNA synthesis from DNA and RNA templates by a viral RNA-dependent RNA polymerase.
In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-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 minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site.
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
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Kao
D001483:Base Sequence; D017795:Bromovirus / Q000235:genetics*; D004279:DNA, Viral; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase