Purification of cowpea mosaic virus RNA replication complex: Identification of a virus-encoded 110,000-dalton polypeptide responsible for RNA chain elongation.
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.
Department of Molecular Biology, Agricultural University, De Dreijen 11, 6703 BC Wageningen, The Netherlands.
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Dorssers
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van der Krol
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J
van der Meer
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A
van Kammen
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