Prototype victorivirus HvV190S employs stop/restart translation to express its RdRp from the downstream ORF in its bicistronic mRNA. The signals for this activity appear to include a predicted RNA pseudoknot directly upstream of the CP stop and RdRp start codons, which overlap in the motif AUGA. Here we used a dual-fluorescence system to further define which HvV190S sequences are important for stop/restart translation and found that the AUGA motif plus 38 nt directly upstream are both necessary and sufficient for this activity. This RNA cassette encompasses the predicted pseudoknot, and indeed substitutions that disrupted the pseudoknot disrupted the activity whereas complementary substitutions that restored the pseudoknot restored the activity. Replacement of this RNA cassette with those from other victoriviruses with a predicted pseudoknot in comparable position also supported stop/restart translation. To our knowledge, this is the first example of stop/restart translation regulated by an RNA pseudoknot.
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