Cytopathogenicity of Classical Swine Fever Virus Correlates with Attenuation in the Natural Host

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Proteolytic processing of polyproteins is considered a crucial step in the life cycle of most positive-strand RNA viruses. An enhancement of NS2-3 processing has been described as a major difference between the noncytopathogenic (non-CP) and the cytopathogenic (CP) biotypes of pestiviruses. The effects of accelerated versus delayed NS2-3 processing on the maturation of the other nonstructural proteins (NSP) have never been compared. In this study, we analyzed the proteolytic processing of NSP in Classical swine fever virus (CSFV). Key to the investigation was a panel of newly developed monoclonal antibodies (MAbs) that facilitated monitoring of all nonstructural proteins involved in virus replication (NS2, NS3, NS4A, NS5A, and NS5B). Applying these MAbs in Western blotting and radioimmunoprecipitation allowed an unambiguous identification of the mature proteins and precursors in non-CP CSFV-infected cells. Furthermore, the kinetics of processing were determined by pulse-chase analyses for non-CP CSFV, CP CSFV, and a CP CSFV replicon. A slow but constant processing of NS4A/B-5A/B occurred in non-CP CSFV-infected cells, leading to balanced lowlevel concentrations of mature NSP. In contrast, the turnover of the polyprotein precursors was three times faster in CP CSFV-infected cells and in cells transfected with a CP CSFV replicon, causing a substantial increase of mature NSP concentrations. We conclude that a delayed processing not only of NS3 but further of all NSP represents a hallmark of regulation in non-CP pestiviruses.

Section: Resultsmentioning

confidence: 99%

Section: Classical Swine Fever Virus (Csfv) Belongs To the Genus Pestmentioning

confidence: 99%

Section: Cells and Viruses Sk-6 Cellsmentioning

confidence: 99%

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Biosynthesis of Classical Swine Fever Virus Nonstructural Proteins

Lamp

Riedel

Roman-Sosa

et al. 2011

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“…The ncp biotype with its downregulated RNA replication efficiency seems to be a prerequisite for efficient control of the innate immune system by the viral virulence factors N pro and E rns as well as for evading induction of apoptosis (9,30). Also for CSFV, it has recently been demonstrated that an upregulation of NS2-3 cleavage correlates with induction of the interferon system and attenuation in the host (14). Therefore, control of NS2-3 cleavage plays a crucial role in the biology of pestiviruses in general.…”

Section: Discussionmentioning

confidence: 99%

Pestivirus Virion Morphogenesis in the Absence of Uncleaved Nonstructural Protein 2-3

Lattwein

Klemens

Schwindt

et al. 2012

The family Flaviviridae contains three genera of positive-strand RNA viruses, namely, Flavivirus, Hepacivirus (e.g., hepatitis C virus [HCV]), and Pestivirus. Pestiviruses, like bovine viral diarrhea virus (BVDV), bear a striking degree of similarity to HCV concerning polyprotein organization, processing, and function. Along this line, in both systems, release of nonstructural protein 3 (NS3) is essential for viral RNA replication. However, both viruses differ significantly with respect to processing efficiency at the NS2/3 cleavage site and abundance as well as functional relevance of uncleaved NS2-3. In BVDV-infected cells, significant amounts of NS2-3 accumulate at late time points postinfection and play an essential but ill-defined role in the production of infectious virions. In contrast, complete cleavage of the HCV NS2-3 counterpart has been reported, and unprocessed NS2-3 is not required throughout the life cycle of HCV, at least in cell culture. Here we describe the selection and characterization of the first pestiviral genome with the capability to complete productive infection in the absence of uncleaved NS2-3. Despite the insertion of a ubiquitin gene or an internal ribosomal entry site between the NS2 and NS3 coding sequences, the selected chimeric BVDV-1 genomes gave rise to infectious virus progeny. In this context, a mutation in the N-terminal third of NS2 was identified as a critical determinant for efficient production of infectious virions in the absence of uncleaved NS2-3. These findings challenge a previously accepted dogma for pestivirus replication and provide new implications for virion morphogenesis of pestiviruses and HCV.

“…All cells were maintained at 37°C and 5% CO 2 . ncp CSFV, cp CSFV-JIV, and a cp CSFV replicon were generated by transfection of a SP6 transcript of p447, p447-JIV (22), and p447-rep cDNA (18) clones as described previously (23). Nucleotide and amino acid numbers of CSFV throughout this study refer to the sequence of the parental ncp CSFV strain Alfort-Tuebingen (GenBank J04358.2).…”

Section: Lassical Swine Fever Virus (Csfv) Is the Causative Agent Omentioning

confidence: 99%

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

Lamp

Riedel

Wentz

et al. 2013

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...