TY - JOUR
T1 - Functional characterization of bovine viral diarrhea virus nonstructural protein 5A by reverse genetic analysis and live cell imaging
AU - Isken, Olaf
AU - Langerwisch, Ulrike
AU - Schönherr, Robert
AU - Lamp, Benjamin
AU - Schröder, Kristin
AU - Duden, Rainer
AU - Rümenapf, Tillmann H.
AU - Tautz, Norbert
PY - 2014/1/1
Y1 - 2014/1/1
N2 - 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 NS5AmCherry to directly visualize functional viral replication complexes by live cell imaging.
AB - 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 NS5AmCherry to directly visualize functional viral replication complexes by live cell imaging.
UR - http://www.scopus.com/inward/record.url?scp=84890889159&partnerID=8YFLogxK
U2 - 10.1128/JVI.01957-13
DO - 10.1128/JVI.01957-13
M3 - Journal articles
C2 - 24131714
AN - SCOPUS:84890889159
VL - 88
SP - 82
EP - 98
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 1
ER -