Key result
The addition of a nontemplated nucleotide at the 3' end of minus-strand brome mosaic virus RNAs led to specific initiation of genomic plus-strand RNA synthesis in vitro.
The study identifies specific nucleotide requirements at the 3' end of minus-strand templates for the initiation of genomic plus-strand RNA synthesis by BMV RNA-dependent RNA polymerase.
Informs in vitro models of viral RNA initiation; leaves open in vivo relevance and extension to other viruses.
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.
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Sivakumaran et al. (1999) studied Brome mosaic virus (BMV) infection (in vitro model). Addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs vs. Cucumber mosaic virus minus-strand RNA templates was evaluated on Initiation of genomic plus-strand RNA synthesis. The addition of a nontemplated nucleotide at the 3' end of minus-strand brome mosaic virus RNAs led to specific initiation of genomic plus-strand RNA synthesis in vitro.
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