Document Detail


Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase.
MedLine Citation:
PMID:  23986594     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Classical swine fever virus (CSFV) is a positive-stranded RNA virus belonging to the genus Pestivirus within the Flaviviridae family. Pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (NS3) that also harbors helicase and NTPase activities essential for RNA replication. In CSFV-infected cells, NS3 appears as two forms, a fully processed NS3 of 80 kDa and the precursor molecule NS2-3 of 120 kDa. Here we report the identification and mapping of additional autocatalytic intramolecular cleavages. One cleavable peptide bond occurs between Leu1781 and Met1782, giving rise to a helicase subunit of 55 kDa and, depending on the substrate, a NS2-3 fragment of 78 kDa (NS2-3p) or a NS3 protease subunit of 26 kDa (NS3p). In trans-cleavage assays using NS4-5 as a substrate, NS3p acts as a fully functional protease that is able to process the polyprotein. NS3p comprises the minimal essential protease, as deletion of Leu1781 results in inactivation. A second intramolecular cleavage was mapped to the Leu1748/Lys1749 peptide bond that yields a proteolytically inactive NS3 fragment. Deletion of either of the cleavage site residues resulted in a loss of RNA infectivity, indicating the functional importance of amino acid identity at the respective positions. Our data suggest that internal cleavage within the NS3 moiety is a common process that further extends the functional repertoires of the multifunctional NS2-3 or NS3 and represents another level of the complex polyprotein processing of Flaviviridae.
Authors:
Benjamin Lamp; Christiane Riedel; Eveline Wentz; Maria-Alejandra Tortorici; Till Rümenapf
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2013-08-28
Journal Detail:
Title:  Journal of virology     Volume:  87     ISSN:  1098-5514     ISO Abbreviation:  J. Virol.     Publication Date:  2013 Nov 
Date Detail:
Created Date:  2013-10-04     Completed Date:  2013-11-27     Revised Date:  2014-05-07    
Medline Journal Info:
Nlm Unique ID:  0113724     Medline TA:  J Virol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  11872-83     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Line
Classical swine fever virus / enzymology*,  physiology*
DNA Mutational Analysis
Hydrolysis
Peptide Hydrolases / metabolism*
Protein Processing, Post-Translational
RNA Helicases / metabolism*
Serine Endopeptidases / metabolism
Viral Nonstructural Proteins / metabolism*
Virus Replication*
Chemical
Reg. No./Substance:
0/NS3 protein, flavivirus; 0/Viral Nonstructural Proteins; EC 3.4.-/Peptide Hydrolases; EC 3.4.21.-/Serine Endopeptidases; EC 3.6.4.13/RNA Helicases
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