Improvements of the infectivity of in vitro transcripts from cloned cowpea mosaic virus cDNA: impact of terminal nucleotide sequences.

Full-length DNA copies of both B- and M-RNA of cowpea mosaic virus (CPMV) were constructed downstream from a T7 promoter. By removal of nucleotides from the promoter sequence, B- and M-RNA-like transcripts with varying numbers of additional nonviral sequences at the 5' end were obtained upon transcription with T7 RNA polymerase. The infectivity of the transcripts in cowpea protoplasts was greatly affected by only a few extra nonviral nucleotides at the 5' end. The addition of about 400 nonviral nucleotides at the 3' end did not have any effect. Using the most infectious transcripts, in 40% of the cowpea protoplasts replication and expression of B-RNA like transcripts were observed and in 10% of the protoplasts both B- and M-RNA-like transcripts multiplied. Moreover, cowpea plants could also be infected with these transcripts. Sequence analysis showed that the 5' terminus of the M-RNA transcripts and the 3' terminus of the B-RNA transcripts were completely restored during replication in plants, including a poly(A) tail of variable length. Swapping experiments have been used to identify an influential point mutation in the coding region for the viral polymerase of a noninfectious B transcript. This experiment demonstrates the potential of the optimized infection system for future analysis of virus-encoded functions.