Viral Evolution and Interferon Resistance of Hepatitis C Virus RNA Replication in a Cell Culture Model

Abstract

Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region. A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-␤ production and low-level IFN-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-␣ treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation. Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly. These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype.Hepatitis C virus (HCV) is a global public health threat that persistently infects an estimated 2% of the world population (46). Although initial HCV infection is usually subclinical, damage accumulates over time in the liver and can result in the development of cirrhosis and end-stage liver disease that often includes hepatocellular carcinoma (37). The virus is a member of the Flaviviridae and contains a 9.6-kb single-stranded positive-sense RNA genome that encodes one large polyprotein whose translation is mediated through an internal ribosome entry site (IRES) found within the viral 5Ј nontranslated region (5Ј NTR). The HCV polyprotein is postranslationally cleaved into at least 10 mature proteins through host peptidase and viral protease activities (35). The HCV nonstructural (NS) proteins are sufficient to support viral replication (4,31,32). HCV RNA replication proceeds in association with intracellular membranes through a viral replicase that includes the NS proteins. The HCV replicase is particularly dependent on the enzymatic activities of the NS5B RNA-dependent RNA polymerase (RdRp) (5) and the NS3/NS4A protease-helicase (reviewed in reference 35). Like other RNA viruses, the replicase of HCV is error prone due to the lack of proofreading function of the NS5B RdRp. Because of this error-prone replication and an overall high replication rate, HCV infection often involves genetically ...

Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling

“…Huh7.5 cells (a gift from C. Rice, The Rockefeller University, New York) are a subline of Huh7 and are highly permissive for HCV RNA replication (8). Huh7-HP, Huh7-A7, and Huh7-K2040 are Huh7 cells that harbor culture-adapted variants of the Con1 HCV (genotype 1b) subgenomic replicon RNA and have been described in detail (3,6,9). Huh7 2-3 and Huh7 2-3c are Huh7-derived cell lines that harbor the full-length self-replicating HCV genome (derived from the HCV-N strain, genotype 1b) and their IFN-cured counterparts, respectively (3,10,11).…”

“…NS3͞4A expression was controlled by culturing cells in the presence (to repress NS3͞4A expression) or absence of tetracycline (to induce NS3͞4A expression) as presented elsewhere (12). Culture methods have been described (3,6). Cell treatments were conducted by replacing culture medium with medium containing 10 ng of IL-1 (R & D Systems), the indicated units͞ml IFN-␣2a (PBL, Piscataway, NJ) or 10 M SCH6 (a gift from Schering-Plough) (3).…”

“…HCV controls induction of the antiviral defense response through the activity of its NS3͞4A protease complex, which disrupts intracellular signaling processes that confer IRF-3 activation (3). Viral control of IRF-3 limits IFN expression to thereby support viral persistence and impart fitness to HCV (3,6). However, the intracellular signaling pathway(s) targeted by NS3͞4A to govern IRF-3 function and HCV replication are incompletely defined.…”

Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity

“…amplified by RT-PCR from the RNA of HCV replicon cell line K2040 (19) and then cloned into pcDNA3 in frame with an N-terminal Flag or Myc tag. The S139A mutant of NS3͞4A and the Cys mutants of MAVS (C435R, C452R, and C508R) were generated by site-directed mutagenesis using the QuikChange kit (Stratagene).…”

Hepatitis C databases, principles and utility to researchers

“…26 Two studies by the group of Peter Simmonds in Edinburgh relied partly on the Los Alamos database: one analyzing RNA secondary structures in the HCV Core and NS5B genes, 27 the other reviewing current knowledge about HCV evolution. 28 Other examples are papers studying a potential role of CD81 as a cellular HCV receptor, 29 the influence of interferon on HCV evolution, 30 and the variability of genotype 4 sequences from South-Western France. 31 …”