Mobility analysis of an NS5A–GFP fusion protein in cells actively replicating hepatitis C virus subgenomic RNA

Abstract

We have introduced GFP and photoactivatable GFP into the NS5A coding region of a hepatitis C virus (HCV) subgenomic replicon that gives efficient transient replication. NS5A-GFP, expressed by the replicon, could be detected in cytoplasmic fluorescent foci as early as 4 h after RNA was introduced into cells. The fluorescent foci are likely to be sites where RNA synthesis could occur, although their production was not dependent on prior replication. Photobleaching studies demonstrated that the fluorescent proteins were relatively immobile upon expression from replicon RNAs. By contrast, an NS5A-GFP chimera produced in the absence of other viral proteins was mobile. Hence, interactions in cells expressing HCV replication proteins limit NS5A mobility, and transfer of viral proteins between foci is either slow or does not occur. Thus, the sites of HCV RNA replication possibly have a fixed complement of proteins that may act as discrete factories for producing viral RNA.Subgenomic replicons (SGRs) of hepatitis C virus (HCV), based on sequences from genotype 1a and 1b strains, have been invaluable for examining viral RNA replication (Blight et al., 2000;Lohmann et al., 1999). Recent reports have demonstrated that SGRs possessing sequences from JFH1, an HCV genotype 2a strain, replicate far more efficiently than those from genotype 1b in both colony-forming assays and quantitative transient replication assays (TRAs) using reporters such as luciferase activity (Kato et al., 2003;Miyamoto et al., 2006;Targett-Adams & McLauchlan, 2005;Windisch et al., 2005). Importantly, efficient replication with JFH1 SGRs is not dependent on either highly permissive Huh-7 cells or tissue-culture-adapted mutations that are needed to enhance genotype 1b replicon competence (Krieger et al., 2001;Lohmann et al., 2001). By contrast to genotype 1b SGRs, the JFH1-based replicon exhibits large increases in luciferase activity over the initial 24 h following transfection of Huh-7 cells with SGR RNA (Targett-Adams & McLauchlan, 2005). Hence, this replicon offers unique possibilities to examine the early stages of viral RNA replication.Previous studies have reported stable cell lines that support steady-state replication of SGRs containing genotype 1b sequences that incorporate GFP-tagged NS5A (Liu et al., 2006; McCormick et al., 2006b;Moradpour et al., 2004). However, replication in TRAs with such replicons was barely detectable (Appel et al., 2005b). Here, we describe development of JFH1-based SGRs that incorporate GFP and a photoactivatable variant of GFP (PAGFP) (Patterson & Lippincott-Schwartz, 2002 into the C-terminal region of the NS5A protein.To construct tagged JFH1 SGRs, the GFP and PAGFP open reading frames were inserted into the 39 coding sequence of JFH1-derived NS5A at a site previously shown to be capable of accommodating GFP within genotype 1b-based replicons (Liu et al., 2006; McCormick et al., 2006b;Moradpour et al., 2004). DNA fragments encoding NS5A-GFP/PAGFP fusion proteins were engineered into pSGR-Luc-JFH1 (TargettAdams & McL...

Three-Dimensional Architecture of Tick-Borne Encephalitis Virus Replication Sites and Trafficking of the Replicated RNA

“…Previous FRAP studies performed with components of the HCV replication complex NS4B and NS5A showed that the internal architecture of the membranous web is relatively static, with limited exchange of viral nonstructural proteins between neighboring factories (17,18,54). In the present study, for the first time, we monitored viral RNA dynamics within perinuclear regions of virus replication.…”

“…In order to provide a global picture of the spatiotemporal organization of the RCs, it is therefore important to integrate high-resolution imaging approaches with innovative techniques that allow exploring in real time the dynamic interplay between viruses and their hosts. Engineered subgenomic replicons expressing fluorescently tagged nonstructural proteins (16)(17)(18) have been exploited to investigate the distribution and dynamics of HCV replicase proteins. However, the only available tool to explore flaviviral RNA trafficking has been reported for TBEV (19).…”

Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1

“…Corresponding constructs (SGR-Luc-JFH1 wt/+1 and SGR-Luc-JFH1 wt/21 ) were also constructed for wt SGR-Luc-JFH1. Luciferase activity measured at 4 h after electroporation of RNA into cells, corresponding to a period before the onset of replication and indicative of translation from input RNA (Jones et al, 2007), gave enzyme levels for SGR-Luc-JFH1 poly(A)/+1 and SGR-Luc-JFH1 poly(A)/21 that were 23 and 9.7% of those obtained for SGR-Luc-JFH1 poly(A) (Fig. 5a).…”

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

“…CMPD (7) exhibits selective inhibition of PI4KIIIa (IC 50 PI4KIIIa: 7 nM, PI4KIIIb: 1.8 µM), whereas CMPD (3) exhibits a similar selectivity to PIK93 (IC 50 PI4KIIIa: 7.3 µM, PI4KIIIb: 15 nM). As a positive control for inhibition of PI4KIIIa we utilized Huh7.5 cells transiently expressing an HCV subgenomic replicon (SGR-Luc-GFP-JFH1), derived from the JFH-1 infectious clone and containing an insertion of GFP into domain III of NS5A (Jones et al, 2007). This allowed HCV genome replication to be assayed using the IncuCyte system, as described for FMDV above.…”

“…Replicons have also been used to study the replication of a number of other viruses. Here, we have also used hepatitis C virus (HCV) sub-genomic replicons pSGR-Luc-GFP-JFH-1 (Jones et al, 2007) and SGR-feo-JFH-1 (Wyles et al, 2009), together with a Coxsackievirus B3 (CVB3) sub-genomic replicon, pRib-Fluc-CB3/T7 (Lanke et al, 2009). …”