Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active. Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, 110K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A).oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.