Three discontinuous loop nucleotides in the 3′ terminal stem-loop are required for Red clover necrotic mosaic virus RNA-2 replication

The genome of Red clover necrotic mosaic virus (RCNMV) consists of positive-sense, single-stranded RNA-1 and RNA-2. The 29 nucleotides at the 3′ termini of both RNAs are nearly identical and are predicted to form a stable stem-loop (SL) structure, which is required for RCNMV RNA replication. Here we performed a systematic mutagenesis of the RNA-2 3′ SL to identify the nucleotides critical for replication. Infectivity and RNA replication assays indicated that the secondary structure of the 3′ SL and its loop sequence UAUAA were required for RNA replication. Single-nucleotide substitution analyses of the loop further pinpointed three discontinuous nucleotides (L1U, L2A, and L4A) that were vital for RNA replication. A 3-D model of the 3′ SL predicted the existence of a pocket formed by these three nucleotides that could be involved in RNA-protein interaction. The functional groups of the bases participating in this interaction at these positions are discussed.