Mutational Analysis ofCis-Acting Sequences in the 3′- and 5′-Untranslated Regions of RNA2 of Red Clover Necrotic Mosaic Virus

Mutational analysis has been used to investigatecis-acting sequences in the 3′- and 5′-untranslated regions of red clover necrotic mosaic virus RNA2 required for replication in the presence of wild-type RNA1. Deletion of a sequence near the 3′ end of RNA2, which is potentially capable of forming a stable stem-loop structure, abolished the ability of RNA2 to replicate inNicotiana clevelandiiprotoplasts, as judged by the failure to detect production of either the positive or negative RNA strand. A base-paired structure in the stem was shown to be essential for replication, but a mutant RNA2 in which every base of the stem had been altered was able to replicate inN. clevelandiiprotoplasts, to produce lesions on leaves ofVigna unguiculataplants, and to infectN. clevelandiiplants systemically. However, changing three bases in the loop of the potential stem-loop structure abolished the replication of RNA2. Upstream sequences in the 3′-untranslated region of RNA2 were also shown to be important for RNA2 replication. Deletions in the 5′-untranslated region of RNA2 showed that sequences across the whole of this region were needed for production of the positive strand but not for production of the negative strand. Some of thecis-acting sequences required for replication of RNA2 have almost identical counterparts in RNA1, but others appear to be unique to RNA2.