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. 1984 Apr;81(7):1951–1955. doi: 10.1073/pnas.81.7.1951

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

Lambert Dorssers 1,*, Sander van der Krol 1, Jos van der Meer 1, Albert van Kammen 1, Pim Zabel 1,†
PMCID: PMC345414  PMID: 16593443

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-SO4/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.

Keywords: RNA-dependent RNA polymerase (RNA replicase), plant virus RNA replication, RNA-protein complex, immunological blotting, virus-specific antibody

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Selected References

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