Small-Scale Isolation of Viral RNA-Dependent RNA Polymerase from Protoplasts Inoculated with In Vitro Transcripts

Cowpea chlorotic mottle virus (CCMV) replicated in tobacco suspension cell protoplasts inoculated with in vitro transcripts of CCMV RNA1, 2, and 3. CCMV RNA-dependent RNA polymerase (RdRp) isolated from these protoplasts specifically recognized CCMV and Brome mosaic virus (BMV) subgenomic RNA promoters and directed in vitro RNA synthesis in a manner indistinguishable from CCMV RdRp more laboriously isolated from systemically infected cowpea leaves. Omission of CCMV RNA3 from the protoplast inoculum or replacement with in vitro transcripts of BMV RNA3 reduced CCMV (+)-strand RNA1 and 2 accumulation to approximately 1/40 and approximately 1/10, respectively, of the level attained when CCMV RNA3 was present. The absence of CCMV RNA3 did not prevent assembly and isolation of highly active, template-dependent and template-specific CCMV RdRp, which directed synthesis of products identical in size to those of RdRp isolated from protoplasts inoculated with all three CCMV genomic RNAs. These results demonstrate that CCMV RNA1 and 2 are sufficient for CCMV replication and RdRp assembly in tobacco protoplasts. This approach for isolation of functional viral RdRp will be especially useful for viruses for which large quantities of infected tissue are unavailable, such as those with specific tissue tropisms or mutants incapable of systemic movement.