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Sindbis virus RNA polymerase is degraded by the N-end rule pathway

de Groot, Raoul J. and Rümenapf, Tillmann and Kuhn, Richard J. and Strauss, Ellen G. and Strauss, James H. (1991) Sindbis virus RNA polymerase is degraded by the N-end rule pathway. Proceedings of the National Academy of Sciences of the United States of America, 88 (20). pp. 8967-8971. ISSN 0027-8424. PMCID PMC52632. doi:10.1073/pnas.88.20.8967.

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Upon infection of animal cells by Sindbis virus, four nonstructural (ns) proteins, termed nsP1-4 in order from 5' to 3' in the genome, are produced by posttranslational cleavage of a polyprotein. nsP4 is believed to function as the viral RNA polymerase and is short-lived in infected cells. We show here that nsP4 produced in reticulocyte lysates is degraded by the N-end rule pathway, one ubiquitin-dependent proteolytic pathway. When the N-terminal residue of nsP4 is changed by mutagenesis, the metabolic stabilities of the mutant nsP4s follow the N-end rule, in that the half-life of nsP4 bearing different N-terminal residues decreases in the order Met > Ala > Tyr ≥ Phe > Agr. Addition of dipeptides Tyr-Ala, Trp-Ala, or Phe-Ala to the translation mixture inhibits degradation of Tyr-nsP4 and Phe-nsP4, but not of Arg-nsP4. Conversely, dipeptides His-Ala, Arg-Ala, and Lys-Ala inhibit the degradation of Arg-nsP4 but not of Tyr-nsP4 or Phe-nsP4. We found that there is no lysine in the first 43 residues of nsP4 that is required for its degradation, indicating that a more distal lysine functions as the ubiquitin acceptor. Strict control of nsP4 concentration appears to be an important aspect of the virus life cycle, since the concentration of nsP4 in infected cells is regulated at three levels: translation of nsP4 requires read-through of an opal termination codon such that it is underproduced; differential processing by the virus-encoded proteinase results in temporal regulation of nsP4; and nsP4 itself is a short-lived protein degraded by the ubiquitin-dependent N-end rule pathway.

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Additional Information:© 1991 National Academy of Sciences. Communicated by James Bonner, July 18, 1991. We are grateful to W. R. Hardy for his help and advice during this project, to N. Davidson for help in deriving the equations used to calculate the nsP4 half-lives, and to A. Varshavsky for critical review of the manuscript. This work was supported by Grants Al 10793 and Al 20612 from the National Institutes of Health. R.J.d.G. was supported by a fellowship from the European Molecular Biology Organization (ALTF 280-1988) and T.R. was supported by a fellowship from Deutsche Forschungsgemeinschaft.
Funding AgencyGrant Number
NIHAl 10793
NIHAl 20612
European Molecular Biology Organization (EMBO)ALTF 280-1988
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
Issue or Number:20
PubMed Central ID:PMC52632
Record Number:CaltechAUTHORS:20141217-072916018
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Official Citation:Sindbis virus RNA polymerase is degraded by the N-end rule pathway. R J de Groot, T Rümenapf, R J Kuhn, E G Strauss, and J H Strauss PNAS 1991 88 (20) 8967-8971; doi:10.1073/pnas.88.20.8967
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:52929
Deposited By: Ruth Sustaita
Deposited On:17 Dec 2014 17:10
Last Modified:10 Nov 2021 19:45

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