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Suppressor Mutations That Allow Sindbis Virus RNA Polymerase to Function with Nonaromatic Amino Acids at the N-Terminus: Evidence for Interaction between nsP1 and nsP4 in Minus-Strand RNA Synthesis

Shirako, Yukio and Strauss, Ellen G. and Strauss, James H. (2000) Suppressor Mutations That Allow Sindbis Virus RNA Polymerase to Function with Nonaromatic Amino Acids at the N-Terminus: Evidence for Interaction between nsP1 and nsP4 in Minus-Strand RNA Synthesis. Virology, 276 (1). pp. 148-160. ISSN 0042-6822. http://resolver.caltech.edu/CaltechAUTHORS:20160125-144218760

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Abstract

The alphavirus RNA polymerase, nsP4, invariably has a Tyr residue at the N-terminus. Previously we reported that the N-terminal Tyr residue of nsP4 of Sindbis virus, the type species of the genus Alphavirus, can be substituted with Phe, Trp, or His without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for Met, were lethal or quasilethal. Here we report the identification of two suppressor mutations in nsP4 (Glu-191 to Leu and Glu-315 to Gly, Val, or Lys) and one in nsP1 (Thr-349 to Lys) that allow nsP4 with nonaromatic amino acids at the N-terminus to function at 30°C. The suppressor mutation at nsP4 Glu-315 occurred most frequently. All three suppressor mutations suppressed the effects of Ala, Arg, or Leu at the N-terminus of nsP4 with almost equal efficiency and thus the effect of the suppressing mutation is independent of the nsP4 N-terminal residue. Reconstructed mutants containing nsP1-T349K or nsP4-E315G combined with Ala-nsP4 had a defect in minus-strand RNA synthesis at 40°C. A double mutant containing nsP4-Q191L combined with Ala-nsP4 was unstable and could not be tested for RNA synthesis because it reverted to temperature-independence too rapidly. Combinations of nsP1-T349K or nsP4-E315G with Leu, Arg, His, or any aromatic amino acid at the N-terminus of nsP4 also made the mutant viruses temperature sensitive. The results from this study and from a previous report on the shutoff of minus-strand RNA synthesis at 40°C with the nsP1-A348T mutation in ts11 suggests that the N-terminus nsP4 interacts with nsP1 during initiation of minus-strand RNA synthesis.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1006/viro.2000.0544DOIArticle
http://www.sciencedirect.com/science/article/pii/S0042682200905440PublisherArticle
Additional Information:© 2000 Academic Press. Received April 26, 2000; returned to author for revision May 25, 2000; accepted July 20, 2000. This work was supported by Grant AI 10793 from the NIH.
Funders:
Funding AgencyGrant Number
NIHAI 10793
Record Number:CaltechAUTHORS:20160125-144218760
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160125-144218760
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:63944
Collection:CaltechAUTHORS
Deposited By: Donna Wrublewski
Deposited On:26 Jan 2016 18:49
Last Modified:26 Jan 2016 18:49

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