Repair of the tRNA-Like CCA Sequence in a Multipartite Positive-Strand RNA Virus

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Abstract
The 3′ portions of plus-strand brome mosaic virus (BMV) RNAs mimic cellular tRNAs. Nucleotide substitutions or deletions in the 3′ CCA of the tRNA-like sequence (TLS) affect minus-strand initiation unless repaired. We observed that 2-nucleotide deletions involving the CCA 3′ sequence in one or all BMV RNAs still allowed RNA accumulation in barley protoplasts at significant levels. Alterations of CCA to GGA in only BMV RNA3 also allowed RNA accumulation at wild-type levels. However, substitutions in all three BMV RNAs severely reduced RNA accumulation, demonstrating that substitutions have different repair requirements than do small deletions. Furthermore, wild-type BMV RNA1 was required for the repair and replication of RNAs with nucleotide substitutions. Results from sequencing of progeny viral RNA from mutant input RNAs demonstrated that RNA1 did not contribute its sequence to the mutant RNAs. Instead, the repaired ends were heterogeneous, with one-third having a restored CCA and others having sequences with the only commonality being the restoration of one cytidylate. The role of BMV RNA1 in increased repair was examined.