Analysis of sequences involved in cowpea mosaic virus RNA replication using site-specific mutants

Using a full-length cDNA clone of cowpea mosaic virus (CPMV) B-RNA from which infectious transcripts can be generated, we examined the influence of a sequence of 11 nucleotides, UUUUAUUAAAA, comprising the nucleotides 5883 to 5893 in the 3' noncoding region of B-RNA, on viral RNA replication. This sequence is not only present in B-RNA but also in M-RNA and represents the 7 nucleotides preceding the poly(A) tail and the first four A residues of the poly(A) tail. Replication of B-RNA transcripts derived from a series of mutants in this region was tested in cowpea plants and protoplasts. Only mutant transcripts with minor modifications appeared able to replicate, which indicates that the region has a function in viral RNA replication. In addition, the results suggest the existence of a hairpin loop in this region. Those transcripts with deletions which disturb the putative hairpin structure have decreased specific infectivities. Mutant transcripts reversed stepwise to the wild-type sequence during replication in plants. This observation strengthens the idea that the sequence of 11 nucleotides has a function in viral RNA replication.