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Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants

Rice, Charles M. and Levis, Robin and Strauss, James H. and Huang, Henry V. (1987) Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. Journal of Virology, 61 (12). pp. 3809-3819. ISSN 0022-538X. http://resolver.caltech.edu/CaltechAUTHORS:RICjvir87

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Abstract

We constructed full-length cDNA clones of Sindbis virus that can be transcribed in vitro by SP6 RNA polymerase to produce infectious genome-length transcripts. Viruses produced from in vitro transcripts are identical to Sindbis virus and show strain-specific phenotypes reflecting the source of RNA used for cDNA synthesis. The cDNA clones were used to confirm the mapping of the causal mutation of ts2 to the capsid protein. A general strategy for mapping Sindbis virus mutations is described and was used to identify two lethal mutations in an original full-length construct which did not produce infectious transcripts. An XbaI linker was inserted in the cDNA clone near the transcriptional start of the subgenomic mRNA; the resulting virus retains the XbaI recognition sequence, thus providing formal evidence that viruses are derived from in vitro transcripts of cDNA clones. The potential applications of the cDNA clones are discussed.


Item Type:Article
Additional Information:Copyright © 1987 by the American Society for Microbiology. Received 3 June 1987/Accepted 25 August 1987 We thank Arash Grakoui and Edith Lenches for expert technical assistance; Steve A. Chervitz for participation in transcription experiments of supercoiled plasmid DNA; Chang S. Hahn and W. Reef Hardy for providing the ts2 cDNA clone and antisera to the nonstructural proteins, respectively; Vince Cannistraro and David Kennell for valuable advice on synthesis of [5'-32P]pCp and 3'-end labeling of RNAs; and Milton Schlesinger and Sondra Schlesinger for helpful suggestions on the manuscript. This work was supported by a Biomedical Research Support grant; Public Health Service grants A124134, A120612, and A110793 from the National Institutes of Health; grant DMB86-17372 from the National Science Foundation; a grant from the Pew Memorial Trust; and the Monsanto/Washington University Biomedical Research Contract. C.M.R. is a Pew Scholar in the Biomedical Sciences.
Record Number:CaltechAUTHORS:RICjvir87
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:RICjvir87
Alternative URL:http://jvi.asm.org/cgi/content/abstract/61/12/3809
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6259
Collection:CaltechAUTHORS
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Deposited On:29 Nov 2006
Last Modified:26 Dec 2012 09:19

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