Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase

Abstract

bClassical 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 Leu 1781 and Met 1782 , 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 transcleavage 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 Leu 1781 results in inactivation. A second intramolecular cleavage was mapped to the Leu 1748 /Lys 1749 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. C lassical swine fever virus (CSFV) is the causative agent of an important disease in pigs and is listed by the World Organization for Animal Health (OIE). CSFV, together with bovine viral diarrhea virus (BVDV) and border disease virus (BDV), represents the genus Pestivirus within the Flaviviridae (1).The genome of CSFV, a positive-stranded RNA molecule of about 12.3 kb, encodes a single polyprotein of 3,899 amino acids that is co-and posttranslationally processed into 12 mature proteins by two cellular and three viral proteases (Npro, nonstructural protein 2 [NS2], and NS3) (2, 3). Npro and NS2 are autoproteases that mediate a single cis-cleavage each. The chymotrypsin-like serine protease NS3 acts in both cis-cleavage (NS3-4A) and trans-cleavage (NS4A-4B-5A-5B) (4). In addition to its protease activity, NS3 mediates helicase (5) and NTPase (6) activities. While the C-terminal DEXH helicase (superfamily 2) shares a high degree of conservation within the members of Flaviviridae, the NS3 serine proteases are variable in sequence and substrate specificity. NS3-mediated cleavages in pestiviruses occur at Leu/Ser, Leu/Ala, and Leu/Asn sites (7), while NS3 of hepatitis C virus (HCV) prefers Cys/X motives (8) and Flavivirus NS3 dibasic motives (9). The NS3 protease of pestiviruses and hepaciviruses utilizes NS4A as an essential cofactor (4), while the NS3 protease of flaviviruses requires NS2B to form the active enzyme (9, 10). Different in vitro models hav...

The Molecular Biology of Pestiviruses

“…All sites downstream of the NS2/NS3 site are processed by the NS3 protease (Elbers, Tautz, Becher, R€ umenapf, & Thiel, 1996;Harada, Tautz, & Thiel, 2000;Lamp et al, 2011;Lamp, Riedel, Wentz, Tortorici, & R€ umenapf, 2013;Tautz, Elbers, Stoll, Meyers, & Thiel, 1997;Xu et al, 1997). NS4A functions as a cofactor of the NS3 protease and is at least essential for processing the NS4B/NS5A and the NS5A/ NS5B sites.…”

X-Ray Structure of the Pestivirus NS3 Helicase and Its Conformation in Solution

“…We also modified this construct to express pNS3h2, a 536-residue protein comprising pNS3 (residues 173 to 683) (33). To express full-length pNS3ph, we used the construct described elsewhere (6), which includes the NS3-activating sequence of NS4A (residues 21 to 55) fused to the N terminus of NS3 (residues 1 to 683) through a GSGS linker. As for the pNS3h constructs, the His tag plus the TEV site and the first 8 residues of NS4A were fused to the N and C termini, respectively.…”

“…Since pNS3ph and pNS3p*h exhibit similar RNA unwinding levels (Fig. 1G), we chose to introduce the mutations in the background of the latter construct to avoid possible artifacts due to the autocatalytic activity described for pNS3ph by Lamp et al (6). Both deletion mutants were active and able to support RNA unwinding activity (Fig.…”

“…The protease and helicase domains of NS3 (NS3p and NS3h, respectively) are believed to function at different stages of the replication cycle, although in the case of HCV, the two domains were shown to enhance each other's activities (16,17). NS3 was recently shown to be autoproteolyzed in CSFV-infected cells to release NS3h (6). In addition, NS3 was shown to play other functions in the virus cycle, such as interfering with the innate immune system in the case of HCV (18) and modulating fatty acid synthesis in the case of dengue virus (DENV) (19).…”

“…The polyprotein is co-and posttranslationally processed by cellular and virus genome-encoded proteases to yield the individual mature viral proteins in the order shown in Fig. 1A (2,5,6). Structural studies are available for nonstructural protein 5B (NS5B) (7), E rns (8), E2 (9,10), and N pro (11,12).…”