Insights into the unique characteristics of hepatitis C virus genotype 3 revealed by development of a robust sub-genomic DBN3a replicon

Abstract

Hepatitis C virus (HCV) is an important human pathogen causing 400 000 chronic liver disease-related deaths annually. Until recently, the majority of laboratory-based investigations into the biology of HCV have focused on the genotype 2 isolate, JFH-1, involving replicons and infectious cell culture systems. However, genotype 2 is one of eight major genotypes of HCV and there is great sequence variation among these genotypes (>30 % nucleotide divergence). In this regard, genotype 3 is the second most common genotype and accounts for 30 % of global HCV cases. Further, genotype 3 is associated with both high levels of inherent resistance to direct-acting antiviral (DAA) therapy, and a more rapid progression to chronic liver diseases. Neither of these two attributes are fully understood, thus robust genotype 3 culture systems to unravel viral replication are required. Here we describe the generation of robust genotype 3 sub-genomic replicons (SGRs) based on the adapted HCV NS3-NS5B replicase from the DBN3a cell culture infectious clone. Such infectious cell culture-adaptive mutations could potentially promote the development of robust SGRs for other HCV strains and genotypes. The novel genotype 3 SGRs have been used both transiently and to establish stable SGR-harbouring cell lines. We show that these resources can be used to investigate aspects of genotype 3 biology, including NS5A function and DAA resistance. They will be useful tools for these studies, circumventing the need to work under the biosafety level 3 (BSL3) containment required in many countries.

Keywords: NS5A; genotype 3; hepatitis C virus; sub-genomic replicon.

Fig. 1.

Structure of the DBN3a infectious clone and bicistronic SGRs. (a) Genome of the DBN3a_cc infectious clone with yellow circles indicating sites of mutation to enhance virus replication and propagation [12]. (b) Schematic of the DBN3a SGR: core, E1, E2, p7 and NS2 coding sequences have been removed, leaving only the 5′ end of the core sequence, coding for the N-terminal 12 amino acids. (ΔCore). The reporter (CpG/UpA-low firefly luciferase, neomycin phosphotransferase or eGFP) is thus expressed as a fusion with the N-terminus of core and is under the translational control of the HCV IRES. The HCV replicase, NS3-NS5B with DBN3a_cc adaptive mutations, is under the translational control of the EMCV IRES.

Fig. 2.

Transient replication of DBN3a-derived SGRs. (a) Huh7.5 cells were electroporated with in vitro transcripts of JFH-1 [10], S52 [9, 18] and DBN3a SGRs containing a CpG/UpA low luciferase, and replication was monitored by measuring the production of luciferase at 4, 24, 48 and 72 h p.e. GND/GNN: polymerase-inactive negative controls. n=3, error bars represent sem. (b) JFH-1 and DBN3a SGRs containing an eGFP reporter were transfected into Huh7.5 cells with corresponding GND/GNN-negative controls. Replication was monitored hourly for 96 h by measuring eGFP expression using an IncuCyte Zoom. n=2, error bars represent sem. (c) Images of JFH-1 and DBN3a eGFP SGR-transfected cells at 24 or 72 h p.e., respectively.

Fig. 3.

Sub-cellular localization of JFH-1 and DBN3a NS5A. (a) Huh7.5 cells were electroporated with SGR-neo-DBN3a RNA. SGR-harbouring cells were selected using G418. Cells were stained with an antibody to NS5A and visualized using immunofluorescent microscopy. (b) SGR-feo-JFH-1- or SGR-neo-DBN3a-harbouring cells were stained with an antibody to NS5A and visualized with confocal microscopy.

Fig. 4.

Phosphorylation of DBN3a NS5A. Lysates of Huh7 or Huh7.5 cells stably harbouring either SGR-feo-JFH-1 or SGR-neo-DBN3a were probed using a sheep polyclonal antiserum to detect total NS5A (red), together with phospho-specific antibodies to pS225 (a) or pS232 (b) (green signal). NS5A species are identified by either red dots (basally phosphorylated) or green dots (hyper-phosphorylated). Naïve Huh7.5 cells were included as a control.

Fig. 5.

Replication of SGR-luc-DBN3a with resistance-associated substitutions (RAS). Huh7.5 cells were electroporated with in vitro-transcribed SGR-luc-DBN3a RNA with either the RAS Y93H or an in-frame deletion of proline 32 (ΔP32). Luciferase levels were measured at the indicated times and compared to mock, original and NS5B-GNN controls. n=3, error bars represent sem. **P <0.01, ***P <0.001 for 72 h p.e. values compared to original.

Fig. 6.

Response of SGR-luc-DBN3a to antiviral chemotherapeutics. Huh7.5 cells were electroporated with in vitro-transcribed SGR-luc-JFH-1 or SGR-luc-DBN3a (original or Y93H) RNA. Compounds were added at the indicated concentrations. Luciferase levels were measured at 72 h p.e. and the effective concentration 50 % (EC₅₀) values were calculated using GraphPad Prism. n=3, error bars represent sem. DCV, daclatasvir; VEL, velpatasvir; SOF, sofosbuvir; CsA, cyclosporin A.