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Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis

Shirako, Yukio and Strauss, James H. (1994) Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis. Journal of Virology, 68 (3). pp. 1874-1885. ISSN 0022-538X. PMCID PMC236650. https://resolver.caltech.edu/CaltechAUTHORS:20160404-164749438

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Abstract

Nonstructural proteins of Sindbis virus, nsPl, nsP2, nsP3, and nsP4, as well as intermediate polyproteins, are produced from two precursor polyproteins, Pl23 and Pl234, by a proteolytic enzyme encoded in the C-terminal half of nsP2. We studied the requirements for and the functions of the intermediate and mature processing products for Sindbis virus RNA synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various combinations. A mutant defective in cleaving both the 1/2 and 2/3 sites, which makes only uncleavable Pl23 and mature nsP4 as final products, produced 10-3 as much virus as did the wild-type virus after 10 hat 30°C and was nonviable at 40°C. A mutant defective in processing the 2/3 site, which makes nsPl, nsP4, and P23 as well as precursor Pl23, grew 10-1 as efficiently as wild-type virus at 30°C and 10-3 as efficiently at 40°C. Early minus-strand RNA synthesis by these mutants was as efficient as that by wild-type virus, whereas plus-strand RNA synthesis was substantially decreased compared with that by wild-type virus. A mutant defective in processing the 3/4 site was nonviable at either 30 or 40°C. The 3/4 site mutant could be complemented by the mutant unable to cleave either the 1/2 or 2/3 site, which can provide mature nsP4. We interpret these results to signify that (i) mature nsP4 is required for RNA replication, (ii) nsP4 and uncleaved Pl23 function in minus-strand RNA synthesis, and (iii) cleavage of Pl23 is required for efficient plus-strand RNA synthesis. We propose that Sindbis virus RNA replication is regulated by differential proteolysis of Pl23. Early in infection, nsP4 and uncleaved Pl23 form transient minus-strand RNA replication complexes which vanish upon cleavage of Pl23. Later in infection, an elevated level of viral proteinase activity eliminates de novo synthesis of Pl23, and no further synthesis of minus-strand RNA is possible. In contrast, nsP4 and cleavage products from Pl23 form plus-strand RNA replication complexes which are stable and remain active throughout the infection cycle.


Item Type:Article
Related URLs:
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http://jvi.asm.org/content/68/3/1874.longPublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC236650/PubMed CentralArticle
Additional Information:© 1994 American Society for Microbiology. Received 20 September 1993/Accepted 7 December 1993. We thank Raoul de Groot for the gift of the 3/4 site mutants and Ellen Strauss for valuable comments on the manuscript. This work was supported by grant AI10793 from the NIH.
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Funding AgencyGrant Number
NIHAI10793
Issue or Number:3
PubMed Central ID:PMC236650
Record Number:CaltechAUTHORS:20160404-164749438
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160404-164749438
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:65912
Collection:CaltechAUTHORS
Deposited By: Donna Wrublewski
Deposited On:05 Apr 2016 16:43
Last Modified:03 Oct 2019 09:51

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