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Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo

de Groot, Raoul J. and Hardy, W. Reef and Shirako, Yukio and Strauss, James H. (1990) Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. EMBO Journal, 9 (8). pp. 2631-2638. ISSN 0261-4189. PMCID PMC552296. https://resolver.caltech.edu/CaltechAUTHORS:20160620-160958379

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Abstract

The non-structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2-containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. As a substrate, we used a polyprotein in which the nsP2 proteinase had been inactivated by a single amino acid substitution. All nsP2-containing polyproteins cleaved the nsP1/2 site in trans. However, proteinases containing nsP1 were unable to cleave the nsP2/3 site. Furthermore, only proteinases containing nsP3 could cleave the nsP3/4 site. These differences in cleavage site specificity result in a temporal regulation of processing in vivo. At 1.7 h post infection P123 and nsP4 accumulated and only small amounts of P34 were found. However, at 4 h post infection P123 was processed rapidly and P34 was produced rather than nsP4. Since nsP4 is thought to be the viral RNA polymerase, the temporal regulation of the nsP4/P34 ratio may be responsible for the temporal regulation of RNA synthesis.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC552296/PubMed CentralArticle
Additional Information:© Oxford University Press. Received on February 20, 1990; revised on April 27, 1990. We thank Ellen G.Strauss for helpful comments and for assistance in the preparation of the manuscript. We thank C. M. Rice for supplying the plasmid pToto1000.S, Randy Levinson for generously providing the plasmid pToto.Cys481-Gly and Richard Kuhn for stimulating discussions and for supplying the plasmid pTotoS7. Furthermore, we thank Alexander van der Bliek for helpful suggestions concerning the PCR experiments and Michael Harrington for advice on densitometry. This work has been supported by Grants AI10793 and AI20612 from the National Institutes of Health and by Grant DMB8617372 from the National Science Foundation. R. J. de Groot was supported by a fellowship of the European Molecular Biology Organization (ALTF 280-1988).
Funders:
Funding AgencyGrant Number
NIHAI-10793
NIHAI-20612
NSFDMB-8617372
European Molecular Biology Organization (EMBO)ALTF 280-1988
Subject Keywords:alphavirus/non-structural proteins/polyprotein/proteinase
Issue or Number:8
PubMed Central ID:PMC552296
Record Number:CaltechAUTHORS:20160620-160958379
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160620-160958379
Official Citation:De Groot RJ, Hardy WR, Shirako Y, Strauss JH. Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. The EMBO Journal. 1990;9(8):2631-2638.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:68553
Collection:CaltechAUTHORS
Deposited By: Donna Wrublewski
Deposited On:22 Jun 2016 04:40
Last Modified:03 Oct 2019 10:14

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