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Identification of the active site residues in the nsP2 proteinase of sindbis virus

Strauss, Ellen G. and de Groot, Raoul J. and Levinson, Randy and Strauss, James H. (1992) Identification of the active site residues in the nsP2 proteinase of sindbis virus. Virology, 191 (2). pp. 932-940. ISSN 0042-6822. PMCID PMC7131396. doi:10.1016/0042-6822(92)90268-T.

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The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.

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Additional Information:© 1992 Academic Press, Inc. Received July 17, 1992; accepted August 19, 1992. This work was supported by Grants AI 10793 and AI 20612 from the National Institutes of Health. R.J. de G. was supported by a fellowship from European Molecular Biology Organization (ALTF 280-1988).
Funding AgencyGrant Number
NIHAI 10793
NIHAI 20612
European Molecular Biology Organization (EMBO)ALTF 280-1988
Issue or Number:2
PubMed Central ID:PMC7131396
Record Number:CaltechAUTHORS:20160203-144803889
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Official Citation:Ellen G. Strauss, Raoul J. De Groot, Randy Levinson, James H. Strauss, Identification of the active site residues in the nsP2 proteinase of sindbis virus, Virology, Volume 191, Issue 2, December 1992, Pages 932-940, ISSN 0042-6822,
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:64207
Deposited By: Donna Wrublewski
Deposited On:04 Feb 2016 01:09
Last Modified:15 Feb 2022 23:37

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