Expression of a cDNA encoding a functional 241-kilodalton vesicular stomatitis virus RNA polymerase

M. Schubert, G. G. Harmison, C. D. Richardson, E. Meier

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Abstract

The large gene, L, of vesicular stomatitis virus (VSV), which codes for the multifunctional RNA-dependent RNA polymerase, was assembled from five overlapping cDNA clones. The sequence of the 6.4-kilobase gene of the final construct was identical to the consensus sequence reported earlier. The gene was inserted into the simian virus 40 transient expression vector pJC119. Antibodies directed against synthetic peptides corresponding to the amino and carboxyl termini of the L protein were raised in rabbits. Both antibodies specifically immunostained the cytoplasm of COS cells that had been transfected with the vector DNA. The expressed L protein was immunoprecipitated from cell extracts and it was identical in size to the L protein of the virion (241 kilodaltons). Most importantly, COS cells that expressed the recombinant L protein transcribed, replicated, and consequently complemented and rescued temperature-sensitive RNA polymerase mutants of VSV at the nonpermissive temperature. The kinetics of virus release were similar to those of a wild-type VSV infection. We conclude that the recombinant RNA polymerase protein L is indistinguishable in its size and functions from the VSV polymerase.

Original languageEnglish
Pages (from-to)7984-7988
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume82
Issue number23
DOIs
Publication statusPublished - 1985
Externally publishedYes

ASJC Scopus Subject Areas

  • General

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Schubert, M., Harmison, G. G., Richardson, C. D., & Meier, E. (1985). Expression of a cDNA encoding a functional 241-kilodalton vesicular stomatitis virus RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America, 82(23), 7984-7988. https://doi.org/10.1073/pnas.82.23.7984