Tagging of NS5A expressed from a functional hepatitis C virus replicon

Abstract

Knowledge of how hepatitis C virus (HCV) proteins associate with components of the host cell to form a functional replication complex is still limited. To address this issue, HCV replicon constructs were generated where either green fluorescent protein (GFP) or the Propionibacterium shermanii transcarboxylase domain (PSTCD) was introduced into the NS5A coding region. Insertion of both GFP and PSTCD was tolerated well, allowing formation of stable replicon-containing cell lines that contained viral protein and transcript levels that were comparable to those of an unmodified parental replicon. Cell lines generated from the GFP-tagged NS5A replicon allowed live-cell visualization of the location of NS5A. Cell lines generated from the PSTCD-tagged replicons allowed rapid and efficient precipitation of the PSTCD-tagged NS5A, as well as other HCV non-structural proteins, using streptavidin-coated magnetic beads. Both replicons represent useful tools that offer different but complementary ways of examining replication-complex formation in cells.Hepatitis C virus (HCV) is an enveloped RNA virus responsible for significant global morbidity and mortality (Lavanchy et al., 1999). In common with other positivestrand RNA viruses, the six non-structural proteins (NS2-NS5B) are likely to form part of a macromolecular complex responsible for replication of the RNA genome. Whilst structural information pertaining to several of the NS proteins is now available (O'Farrell et al., 2003;Tellinghuisen et al., 2005;Yao et al., 1999), very little is known about the structure and composition of the RNA replication complex. Identifying both the viral and cellular components of this complex will prove important, not only in providing an insight into how replication of the viral genome is coordinated, but also in the rational design of therapeutics for treating HCV infection.Progress towards this goal will require the development of robust protocols for isolation of functional RNA replication complexes. This could potentially be achieved by affinity purification of either the viral RNA or the NS proteins. In this regard, RNA replication complexes were recently isolated from replicon-harbouring cells by a two-step purification process: following hybridization with biotinand digoxigenin-tagged oligonucleotides, complexes were purified by sequential avidin-agarose and anti-digoxigenin precipitation steps (Waris et al., 2004). This allowed the isolation of complexes that contained all of the NS proteins and were capable of in vitro RNA synthesis. In a separate study, it was shown that functional HCV replicons could be generated in which either a small epitope tag or the coding sequence for enhanced green fluorescent protein (EGFP) was inserted in frame close to the C terminus of NS5A. The NS5A-EGFP fusion protein could be directly visualized and was used to demonstrate that NS5A co-localized with both NS3 and the sites of viral RNA synthesis (Moradpour et al., 2004;Appel et al., 2005). Based on these studies, we speculated that it might be pos...

Hepatitis C Virus NS2 Protein Serves as a Scaffold for Virus Assembly by Interacting with both Structural and Nonstructural Proteins

“…4C [compare lanes 1 and 2 in the third and fourth panels] and A). These results confirm a prior report of a weak association between NS3 and NS5A demonstrated in pulldown experiments with lysates of cells containing subgenomic replicons expressing NS5A with a PSTCD tag (23). These subgenomic RNAs replicate autonomously but do not produce infectious virus, making it likely that the weak interaction between NS3 and NS5A that we observed here reflects the common presence of the two proteins in the replication complex.…”

Development and Application of Hepatitis C Reporter Viruses with Genotype 1 to 7 Core-Nonstructural Protein 2 (NS2) Expressing Fluorescent Proteins or Luciferase in Modified JFH1 NS5A

“…Various modifications, such as deletions and insertions in NS5A domain III, did not have a major impact on the RNA replication of the Con-1 replicon and of genotype 2a recombinant JFH1 as well as JFH1-based intragenotypic recombinants J6/ JFH1 and Jc1 (4,5,25,28,29,45,46). However, certain sequences of NS5A domain III and especially its C-terminal portion were shown previously to be important for the production of infectious JFH1, J6/JFH1, and Jc1 viruses (5,28,45).…”

Studying Hepatitis C Virus: Making the Best of a Bad Virus

“…A failed attempt at determining an NMR structure for domain II may indicate significant conformational flexibility (127). Domain III appears even more plastic, as this region can tolerate large insertions and deletions without disrupting RNA replication (6,132,146,158). Replicons selected for partial resistance to ribavirin possess mutations in domain III (176).…”