Z Gastroenterol 2010; 48 - P4_06
DOI: 10.1055/s-0029-1246500

The Membrane Anchor of GB virus B Nonstructural Protein 5B Can Substitute the Hepatitis C Virus NS5B Transmembrane Domain: Implications for a Conserved Protein Interaction Site

V Brass 1, J Gouttenoire 2, A Wahl 3, Z Pal 3, HE Blum 3, F Penin 4, D Moradpour 5
  • 1Abteilung Innere Medizin II, Uniklinik Freiburg, Freiburg
  • 2Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland
  • 3Department of Medicine II, University of Freiburg, Freiburg
  • 4Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, Université de Lyon, Lyon, France
  • 5Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Schweiz

Background and aim: Hepatitis C virus (HCV) nonstructural proteins form a membrane-associated replication complex. HCV Nonstructural protein 5B (NS5B), the viral RNA-dependent RNA polymerase (RdRp), is a tail-anchored protein with a highly conserved C-terminal transmembrane domain (TMD) that is required for the assembly of a functional replication complex. The aim of this study was to further validate the function of the NS5B TMD in the process of replication complex formation.

Methodology: Systematic sequence comparisons and predictions of transmembrane segments were coupled with biochemical analyses to identify comparable membrane anchor sequences in HCV related viruses. Full-length RdRps and green fluorescence protein (GFP) fusion constructs were analyzed by confocal laser scanning microscopy and membrane flotation analyses. An HCV-GB virus B (GBV-B) chimeric RdRp was analyzed in the replicon system. Furthermore, sequence analysis of chimeric replicons and directed mutagenesis were performed to search for compensatory mutations.

Results: Here, we describe that the HCV TMD can be functionally replaced by a newly identified analogous membrane anchor of GB virus B NS5B RdRp. Replicons with a chimeric RdRp consisting of the HCV catalytic domain and the GBV-B transmembrane segment replicated with markedly reduced efficiency. Efficient replication of these chimeras was rescued by amino acid changes at a defined position within the transmembrane segment. These observations underline the essential role of the HCV RdRp transmembrane segment for RNA replication and highlight the concept of multifunctionality of membrane anchor segments of HCV and related viruses as possible target for antiviral intervention.