A Y-shaped RNA structure in the 3' untranslated region together with the trans-activator and core promoter of Red clover necrotic mosaic virus RNA2 is required for its negative-strand RNA synthesis.

Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome. RNA1 encodes N-terminally overlapping replication proteins, p27 and p88. RNA2 is replicated efficiently by the replication proteins supplied in trans, whereas RNA1 needs p88 preferentially in cis for its replication. cis-Acting elements required for RNA2 replication have been mapped to the 3' terminal stem-loop structure conserved between RNA1 and RNA2, and to the protein-coding region including the trans-activator. Here, we have identified a Y-shaped RNA structure with three-way RNA junctions predicted in the 3' untranslated region of RNA2 as a novel element required for negative-strand synthesis using an in vitro translation/replication system. We also show that, in addition to the 3' terminal core promoter, several RNA elements including the trans-activator are also required for negative-strand synthesis. Functional roles and structural requirements of these cis-acting elements in RCNMV RNA replication are discussed.