Generation and characterization of a hepatitis C virus NS3 protease-dependent bovine viral diarrhea virus

Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. T o develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding r egion of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 pro tease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B juncti on site and mimicked the proteolytic function of Npro in the release of BVD V core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts de rived from both chimeric clones, P-H/B (wild-type HCV NS3 protease) and P-H /B(S139A) (mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the P-H/B clone yielded viable virus es, whereas the mutant clone, P-H/B(S139A), failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer . Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type an d the chimeric BVDVs. Finally, to assess the genetic stability of the chime ric virus, an Npro-null BVDV (BVDV-Npro in which the entire Npro coding reg ion was deleted) was produced. Although cytopathic, BVDV-Npro was highly de fective in viral replication and growth, a finding consistent with the obse rved stability of the chimeric virus after serial passages.