title: Functional characterization of bovine viral diarrhea virus nonstructural protein 5A by reverse genetic analysis and live cell imaging.
abstract: Nonstructural protein 5A (NS5A) of bovine viral diarrhea virus (BVDV) is a hydrophilic phosphoprotein with RNA binding activity and a critical component of the viral replicase. In silico analysis suggests that NS5A encompasses three domains interconnected by two low-complexity sequences (LCSs). While domain I harbors two functional determinants, an N-terminal amphipathic helix important for membrane association, and a Zn-binding site essential for RNA replication, the structure and function of the C-terminal half of NS5A are still ill defined. In this study, we introduced a panel of 10 amino acid deletions covering the C-terminal half of NS5A. In the context of a highly efficient monocistronic replicon, deletions in LCS I and the N-terminal part of domain II, as well as in domain III, were tolerated with regard to RNA replication. When introduced into a bicistronic replicon, only deletions in LCS I and the N-terminal part of domain II were tolerated. In the context of the viral full-length genome, these mutations allowed residual virion morphogenesis. Based on these data, a functional monocistronic BVDV replicon coding for an NS5A variant with an insertion of the fluorescent protein mCherry was constructed. Live cell imaging demonstrated that a fraction of NS5A-mCherry localizes to the surface of lipid droplets. Taken together, this study provides novel insights into the functions of BVDV NS5A. Moreover, we established the first pestiviral replicon expressing fluorescent NS5A-mCherry to directly visualize functional viral replication complexes by live cell imaging.
authors.affiliation: Institute of Virology and Cell Biology, University of Lübeck, Lübeck, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Isken
authors.forename: Ulrike
authors.initials: U
authors.lastname: Langerwisch
authors.forename: Robert
authors.initials: R
authors.lastname: Schönherr
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lamp
authors.forename: Kristin
authors.initials: K
authors.lastname: Schröder
authors.forename: Rainer
authors.initials: R
authors.lastname: Duden
authors.forename: Tillmann H
authors.initials: TH
authors.lastname: Rümenapf
authors.forename: Norbert
authors.initials: N
authors.lastname: Tautz
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers; D001908:Diarrhea Viruses, Bovine Viral / Q000378:metabolism* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D017931:DNA Primers; D017361:Viral Nonstructural Proteins