Polymerization of nontemplate bases before transcription initiation at the 3′ ends of templates by an RNA-dependent RNA polymerase: An activity involved in 3′ end repair of viral RNAs

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Abstract
The 3′ ends of RNAs associated with turnip crinkle virus (TCV), including subviral satellite (sat)C, terminate with the motif CCUGCCC-3′. Transcripts of satC with a deletion of the motif are repaired to wild type (wt) in vivo by RNA-dependent RNA polymerase (RdRp)-mediated extension of abortively synthesized oligoribonucleotide primers complementary to the 3′ end of the TCV genomic RNA. Repair of shorter deletions, however, are repaired by other mechanisms. SatC transcripts with the 3′ terminal CCC replaced by eight nonviral bases were repaired in plants by homologous recombination between the similar 3′ ends of satC and TCV. Transcripts with deletions of four or five 3′ terminal bases, in the presence or absence of nonviral bases, generated progeny with a mixture of wt and non-wt 3′ ends in vivo. In vitro, RdRp-containing extracts were able to polymerize nucleotides in a template-independent fashion before using these primers to initiate transcription at or near the 3′ end of truncated satC templates. The nontemplate additions at the 5′ ends of the nascent complementary strands were not random, with a preference for consecutive identical nucleotides. The RdRp was also able to initiate transcription opposite cytidylate, uridylate, guanylate, and possibly adenylate residues without exhibiting an obvious preference, flexibility previously unreported for viral RdRp. The unexpected existence of three different repair mechanisms for TCV suggests that 3′ end reconstruction is critical to virus survival.