Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.Pestiviruses are animal pathogens that are recognized as a separate genus of the family Flaviviridae, which also includes the genera Flavivirus and Hepacivirus (hepatitis C viruses [HCV]), as well as the unassigned GB viruses (32). Pestiviruses are widely used as a surrogate model for studying HCV, which grows poorly in available cell culture systems. Persistent HCV infections are a major cause of liver cirrhosis and hepatocellular carcinoma in humans worldwide.The pestiviral genome is a positive-stranded RNA of 12.3 kb. It is translated into a large polyprotein, which is cotranslationally and posttranslationally processed by viral and cellular proteases. The order of proteins in the polyprotein is NH 2 -N pro -C-E rns -E1-E2-p7-NS2-3-NS4A-NS4B-NS5A-NS5B-COOH. The autoprotease N pro generates its C terminus and the N terminus of the downstream core protein C. The proteolytic releases of the structural glycoproteins E rns (RNase secreted), E1, E2, and p7 are mediated by cellular signal peptidases. The nonstructural protein 4A (NS4A)-dependent chymotrypsin-like serine protease in NS3 mediates processing in the NS region downstream of NS3 (32). The mechanism of NS2-3 cleavage was hitherto unknown and is the subject of this study (see below).The pestivirus bovine viral diarrhea virus (BVDV) can establish lifelong persistent infections in animals, which become the primary sources for the horizontal sp...

Polyprotein processing control is a crucial step in the life cycle of positive-strand RNA viruses. Recently, a vital autoprotease generating an essential viral replication factor was identified in such a virus, namely, the pestivirus bovine viral diarrhea virus. Surprisingly, the activity of this protease, which resides in nonstructural protein 2 (NS2), diminishes early after infection, resulting in the limitation of viral RNA replication. Here, we describe that a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) acts as a cofactor of the NS2 protease. Consumption of the intracellular Jiv pool is responsible for temporal regulation of protease activity: overexpression of Jiv interfered with regulation and correlated with increased accumulation of viral RNA; downregulation of the cellular Jiv level accelerated the decline of protease activity and reduced intracellular viral RNA levels and virion production. Accordingly, the amount of a cellular protein controls pestiviral replication by limiting the generation of active viral protease molecules and replication complexes. Importantly, this unique mechanism of replication control is essential for maintenance of the noncytopathogenic phenotype of the virus and thereby for its ability to establish persistent infections. These results add an entirely novel aspect to the understanding of the molecular basis of viral persistence.The Flaviviridae family comprises the genera Flavivirus, Pestivirus, and Hepacivirus; the latter includes the human pathogen Hepatitis C virus (HCV) (13). With an estimated 200 million cases of chronic infections worldwide, HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. Due to their close relationship, pestiviruses, especially bovine viral diarrhea virus (BVDV), represent a widely used surrogate system for HCV.The single-stranded RNA genome of BVDV is of positive polarity and has a length of 12.3 kb. Gene expression occurs via translation of one polyprotein which is processed by cellular and viral proteases giving rise to 12 mature proteins (18). Processing of nonstructural protein 2-3 (NS2-3) into NS2 and NS3 is exerted by a recently characterized vital cysteine autoprotease located in NS2 (17). Interestingly, this enzyme is distantly related to the HCV NS2-3 protease which mediates the analogous cleavage in the HCV polyprotein (11,14,18). The NS4A-dependent chymotrypsin-like serine protease in NS3 catalyzes four processing events in the viral polyprotein (29,32,33). Moreover, NS3 has helicase and NTPase activity (28, 31). The enzymatic functions of NS3 are essential for viral RNA replication which is accomplished by a replication complex (replicase) containing NS3 and four other NS proteins including NS5B, the viral RNA-dependent RNA polymerase (34), as essential constituents (3, 12). The NS2 protease-mediated cleavage of NS2-3 is essential for replication of BVDV, since its cleavage product, NS3, cannot be functionally replaced by NS2-3 in the viral replicase (17). Uncleaved NS2-3 plays a c...

Replication of positive-strand RNA viruses involves translation of polyproteins which are proteolytically processed into functional peptides. These maturation steps often involve virus-encoded autoproteases specialized in generating their own N or C termini. Nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus represents such an enzyme. Bovine viral diarrhea virus NS2 creates in cis its own C terminus and thereby releases an essential viral replication factor. As a unique feature, this enzyme requires for proteolytic activity stoichiometric amounts of a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) or its fragment Jiv90. To obtain insight into the structural organization of the NS2 autoprotease, the basis for its restriction to cis cleavage, as well as its activation by Jiv, we dissected NS2 into functional domains. Interestingly, an N-terminal NS2 fragment covering the active center of the protease, cleaved in trans an artificial substrate composed of a C-terminal NS2 fragment and two downstream amino acids. In the authentic NS2, the 4 Cterminal amino acids interfered with binding and cleavage of substrates offered in trans. These findings strongly suggest an intramolecular product inhibition for the NS2 autoprotease. Remarkably, the chaperone fragment Jiv90 independently interacted with protease and substrate domain and turned out to be essential for the formation of a protease͞substrate complex that is required for cleavage. Thus, the function of the cell-derived protease cofactor Jiv in proteolysis is regulation of protease͞substrate interaction, which ultimately results in positioning of active site and substrate peptide into a cleavage-competent conformation.hepacivirus ͉ J-domain chaperone ͉ pestivirus ͉ protease cofactor T he pestiviruses, together with the flaviviruses and hepatitis C virus, form the family Flaviviridae (1). Replication of these positive-strand RNA viruses involves translation of one polyprotein that is processed by cellular and virus-encoded proteases (2). These processing events generate the proteins required for viral RNA replication and the production of infectious progeny.Recently in nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus (BVDV) an autoprotease was identified (3). In the polyprotein of BVDV strain NCP7, NS2 encompasses amino acids 1137-1589 and thus has a length of 453 aa (4). The N terminus of NS2 is generated by cellular signal peptidases, and the protein remains associated to intracellular membranes, presumably at the endoplasmatic reticulum. The N-terminal half of NS2 is highly hydrophobic and therefore likely to be involved in membrane association. However, so far no experimental data on membrane topology or protein structure are available, in part because of its biochemical properties as well as its toxicity for bacteria. A recent study revealed that NS2 is a cysteine protease (3); the obtained data indicated a catalytic role for histidine at position 1447 (H1447) and cysteine at pos...