Response of Hepatitis C Virus to Long-Term Passage in the Presence of Alpha Interferon: Multiple Mutations and a Common Phenotype

Abstract

H epatitis C virus (HCV) infects an estimated 180 million people worldwide, and about 75% of newly infected patients progress toward a chronic infection, which constitutes a risk for severe liver diseases such as cirrhosis and hepatocarcinoma (1-4). HCV is a hepacivirus in the Flaviviridae family characterized by the error-prone replication and quasispecies dynamics typical of RNA viruses (2,(5)(6)(7). No vaccine is available to prevent HCV infections or disease, and the current standard of care treatment consists of the combination of pegylated alpha interferon (IFN-␣) and the purine nucleoside analogue ribavirin (1-␤-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) (8-10). Type I interferons (IFNs) such as IFN-␣ are members of a family of cytokines that constitute key components of the innate immune response to viruses, and their induction results in an antiviral state of the cell and suppression of viral replication (11)(12)(13)(14). Ribavirin is currently used to treat a number of human viral infections, and it can display multiple mechanisms of action, including enhancement of Th1 antiviral immune responses, upregulation of IFN-stimulated genes (ISGs), depletion of GTP pools, or viral RNA mutagenesis (see reviews in references 15, 16 and 17). However, on average only about 60% of the chronically infected patients show a sustained virological response to treatment with IFN-␣ plus ribavirin that results in virus clearance (18-21). The molecular and physiological bases of the therapeutic activity of the current combination treatment with IFN-␣ plus ribavirin are poorly understood. It might be possible to establish new treatment protocols based on recently developed directly acting antiviral agents (DAAs), with or without .Highly variable viruses exploit a number of strategies to counteract selective constraints intended to prevent their replication (reviewed in reference 7). Mutations that confer resistance to DAAs generally map in the target protein or in a protein that interacts with the target (28-30). However, the complexity of the cellular IFN response pathway is expected to require greater diversity of viral resistance mutations. Current evidence suggests that the response of HCV to IFN-␣-based therapy is influenced by several host (i.e., interleukin-28B [IL-28B] gene polymorphisms) and viral genetic (i.e., viral genotype and population complexity) factors (31-37).The advent of cell culture systems for sustained replication of HCV offers new prospects to approach the evolutionary dynamics of HCV and the host cell-virus relationship, including the anti-HCV activity of IFN-␣. Using these systems, Garaigorta and Chisari documented that HCV-induced protein kinase R (PKR) phosphorylation inhibited translation of IFN-stimulated genes (ISGs) in infected hepatocytes (38). Here we describe the adaptation of the HCV replication system of genotype 2a (39-41) to perform serial passages of HCV in the Huh-7.5 hepatocyte cell line with sustained, efficient viral replication for at least 100 serial passages. This cell ...

Identification of Alpha Interferon-Induced Envelope Mutations of Hepatitis C Virus In Vitro Associated with Increased Viral Fitness and Interferon Resistance

“…1). Supporting this observation, we and others previously showed that IFN-␣ treatment suppressed viral infection in Huh7.5 cells (24,38,40,(45)(46)(47). In addition, in cells from a typical transfection experiment, as shown in Fig.…”

“…13). Finally, Perales et al found that control virus populations serially passaged without IFN-␣ acquired multiple putative cell culture adaptive mutations and showed a certain level of IFN-␣ resistance (40). For the escape experiments done in our study (Fig.…”

“…These genome regions and aspects of the viral life cycle could not be studied in subgenomic replicons, highlighting the importance of the availability of full viral life cycle cell culture systems for studies of interferon resistance of HCV. Earlier this year, a first approach to induce IFN resistance in a full viral life cycle genotype 2a culture system was reported (40). By serial passage in Huh7.5 cells in the presence of up to 10 IU/ml IFN-␣2b but also in the absence of IFN-␣2b, Perales et al were able to select viral populations showing a certain degree of IFN-␣ resistance (40).…”

“…Earlier this year, a first approach to induce IFN resistance in a full viral life cycle genotype 2a culture system was reported (40). By serial passage in Huh7.5 cells in the presence of up to 10 IU/ml IFN-␣2b but also in the absence of IFN-␣2b, Perales et al were able to select viral populations showing a certain degree of IFN-␣ resistance (40). Multiple mutations were observed in viruses passaged with or without IFN-␣.…”

Lipopolysaccharide-induced cytokine expression pattern in peripheral blood mononuclear cells in childhood obesity

“…IFN-α is a soluble cytokine known for its antiviral and immunotherapeutic properties by regulating a diverse set of cytokines (42) and linking innate cell-mediated responses to the adaptive immune response (43). LPS is a natural ligand for the stimulated secretion of IFN-α.…”