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Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123

Shirako, Yukio and Strauss, James H. (1990) Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123. Virology, 177 (1). pp. 54-64. ISSN 0042-6822. doi:10.1016/0042-6822(90)90459-5.

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The cleavage between nsP1 and nsP2 and that between nsP2 and nsP3 in the Sindbis virus nonstructural polyproteins was studied with respect to order of processing and enzyme-substrate relationships, using site-specific mutants in which the cleavage sites had been altered. The penultimate Gly in nsP1 or nsP2 or both was substituted by Ala, Val, or Glu, and processing was studied in vitro. Substitution with Ala resulted in partial cleavage whereas substitution with Val or Glu totally abolished cleavage at the mutagenized site. Abolishment of cleavage at the nsP2/nsP3 site did not affect processing at the nsP1/nsP2 site in the precursor polyprotein P123, and nsP1 and P23 were produced. When cleavage at the nsP1/nsP2 site was abolished, however, processing at the nsP2/nsP3 site was also prevented and P123 accumulated. To investigate why cleavage at the nsP1/nsP2 site should be required for cleavage at the nsP2/nsP3 site, the mutagenized polypeptides were used as enzymes in trans-cleavage experiments. We found that P123 can cleave the nsP1/nsP2 site but not the nsP2/nsP3 site, whereas P23 can cleave the nsP2/nsP3 site very efficiently. Thus, cleavage at the nsP1/nsP2 site by P123 is required to produce an enzyme capable of cleaving the nsP2/nsP3 site. Release of nsP4 from Pi 234 appears to be independent of the other cleavages and occurs primarily immediately after translation. These mutations were also transferred into a full-length cDNA clone of Sindbis virus and virus was recovered. Mutants defective in the cleavage of the nsP2/nsP3 site were temperature sensitive, growing at a slightly reduced rate compared to wild-type virus at 30° but growing poorly at 40°. Mutants defective in the cleavage of both the nsP1/nsP2 site and the nsP2/nsP3 site were viable but grew poorly compared with wild-type at any temperature.

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Additional Information:© 1990 Academic Press. Received January 23, 1990; accepted March 13, 1990. We are grateful to R. de Groot, R. Kuhn, and E. Strauss for stimulating discussions and help in preparation of the manuscript. This work was supported by Grants AI-10793 and AI-20612 from the National Institutes of Health and by Grant DMB86-17372 from the National Science Foundation.
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Official Citation:Yukio Shirako, James H. Strauss, Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123, Virology, Volume 177, Issue 1, 1990, Pages 54-64, ISSN 0042-6822, (
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ID Code:64866
Deposited By: Donna Wrublewski
Deposited On:01 Mar 2016 00:27
Last Modified:10 Nov 2021 23:36

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