Purification of cowpea mosaic virus RNA replication complex: Identification of a virus-encoded 110,000-dalton polypeptide responsible for RNA chain elongation

Abstract

An endogenous cowpea mosaic virus (CPMV) RNA-protein complex (CPMV replication complex) capable of elongating in vitro preexisting nascent chains to full-length viral RNAs has been solubilized from the membrane fraction of CPMV-infected cowpea leaves using Triton X-100 and purified by Sepharose 2B chromatography and glycerol gradient centrifugation in the presence of Triton X-100. Analysis of the polypeptide composition of the complex by NaDod-SO(4)/PAGE and silver staining revealed major polypeptides with molecular masses of 110, 68, and 57 kilodaltons (kDa), among which the 110-kDa polypeptide was consistently found to cosediment precisely with the RNA polymerase activity. Using antisera to specific viral proteins, we found the 110-kDa polypeptide to be the only known viral polypeptide associated with the RNA replication complex, the 68- and 57-kDa polypeptides being most probably host-specific. The host-encoded 130-kDa monomeric RNA-dependent RNA polymerase, which is known to be stimulated in CPMV-infected cowpea leaves, did not copurify with the virus-specific RNA polymerase complex. Our results dispute the hypothesis that plant viral RNA replication may be mediated by the RNA-dependent RNA polymerase of uninfected plants. We tentatively conclude that the 110-kDa polypeptide encoded by the bottom component RNA of CPMV constitutes the core of the CPMV RNA replication complex.