PROTOPLASTS TRANSIENTLY EXPRESSING THE 200K CODING SEQUENCE OF COWPEAMOSAIC VIRUS-B-RNA SUPPORT REPLICATION OF M-RNA

In order to identify the viral polymerase involved in cowpea mosaic vi rus (CPMV) RNA replication the 87K. 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowp ea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that we re indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the exp ression vectors proved that the 24K protease contained within these pr oteins is active. Moreover, it was established that protoplasts transf ected with the expression vector containing the entire 200K coding seq uence, but not those transfected with vectors containing the 170K, 110 K or 87K coding sequences, were able to support replication of co-inoc ulated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specif ic RNA polymerase activity dependent on exogenous added template RNA c ould 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 e xhibited RNA polymerase activity in such assays. These results indicat e that the CPMV polymerase, unlike the poliovirus polymerase, is not a ble to use oligo(U) as a primer or cannot function on exogenous templa te and primer RNA.