RNA REPLICATION MECHANISM OF DIANTHOVIRUS

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Red clover necrotic mosaic virus genomic RNA1 and RNA2 lacked a 5' cap structure, and uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to capped transcripts in cowpea protoplasts. By using a luciferase reporter assay system in vivo, we showed that the 3'-untranslated region (3'-UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5' cap structure. Deletion studies revealed that a stem-loop structure predicted in the 3'-UTR plays an important role in cap-independent translation. The cap-independent translational activity is required for RCNMV RNA1 replication.Translation of RNA2 was associated with RNA2 replication and differed from that of RNA1. Requirement of the formation of RNA replication complex for the translation of RNA2 suggested involvement of replication factors in the RNA2 translation.The stem-loop structure that functions as a trans-activator for the RNA-mediated CP expression is critically required for RNA2 replication itself. However, the RNA-RNA interaction is not required for RNA2 replication and these functions were independent. Nucleotide sequences in the stem-loop of RNA2 also are important for RNA2 replication, suggesting their role on minus-strand RNA possibly in plus-strand RNA synthesis.RCNMV RNA1 had RNA silencing suppressor activity. The activity required RCNMV replicase component proteins and viral RNA which could form viral RNA replication complex with the replicase component proteins. This suggests the involvement of RNA replication factors in RNA silencing suppressing mechanism of RCNMV and also the involvement of RNA silencing factors in RNA replicase complex of RCNMV.