title: Biosynthesis of classical swine fever virus nonstructural proteins.
abstract: 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 low-level 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.
authors.affiliation: Institute of Virology, Faculty of Veterinary Medicine, Frankfurter Strasse 107, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lamp
authors.forename: Christiane
authors.initials: C
authors.lastname: Riedel
authors.forename: Gleyder
authors.initials: G
authors.lastname: Roman-Sosa
authors.forename: Manuela
authors.initials: M
authors.lastname: Heimann
authors.forename: Sylvaine
authors.initials: S
authors.lastname: Jacobi
authors.forename: Paul
authors.initials: P
authors.lastname: Becher
authors.forename: Heinz-Jürgen
authors.initials: HJ
authors.lastname: Thiel
authors.forename: Tillmann
authors.initials: T
authors.lastname: Rümenapf
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000235:genetics* / Q000502:physiology*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D011499:Protein Processing, Post-Translational; D015531:Radioimmunoprecipitation Assay; D013552:Swine; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D017361:Viral Nonstructural Proteins