Functional characterization of bovine viral diarrhea virus nonstructural protein 5A by reverse genetic analysis and live cell imaging

Olaf Isken, Ulrike Langerwisch, Robert Schönherr, Benjamin Lamp, Kristin Schröder, Rainer Duden, Tillmann H. Rümenapf, Norbert Tautz*

*Korrespondierende/r Autor/-in für diese Arbeit
14 Zitate (Scopus)

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 NS5AmCherry to directly visualize functional viral replication complexes by live cell imaging.

OriginalspracheEnglisch
ZeitschriftJournal of Virology
Jahrgang88
Ausgabenummer1
Seiten (von - bis)82-98
Seitenumfang17
ISSN0022-538X
DOIs
PublikationsstatusVeröffentlicht - 01.01.2014

Strategische Forschungsbereiche und Zentren

  • Forschungsschwerpunkt: Infektion und Entzündung - Zentrum für Infektions- und Entzündungsforschung Lübeck (ZIEL)

DFG-Fachsystematik

  • 204-04 Virologie

Fingerprint

Untersuchen Sie die Forschungsthemen von „Functional characterization of bovine viral diarrhea virus nonstructural protein 5A by reverse genetic analysis and live cell imaging“. Zusammen bilden sie einen einzigartigen Fingerprint.

Zitieren