title: Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase.
abstract: 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.affiliation: Institute of Virology, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lamp
authors.forename: Christiane
authors.initials: C
authors.lastname: Riedel
authors.forename: Eveline
authors.initials: E
authors.lastname: Wentz
authors.forename: Maria-Alejandra
authors.initials: MA
authors.lastname: Tortorici
authors.forename: Till
authors.initials: T
authors.lastname: Rümenapf
mesh_terms: D000818:Animals; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000201:enzymology* / Q000502:physiology*; D004252:DNA Mutational Analysis; D006868:Hydrolysis; D010447:Peptide Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020365:RNA Helicases / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D010447:Peptide Hydrolases; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases