Gastroenterology

Gastroenterology

Volume 117, Issue 5, November 1999, Pages 1187-1197
Gastroenterology

Liver, Pancreas, and Biliary Tract
Hepatitis C virus NS5A protein inhibits interferon antiviral activity, but the effects do not correlate with clinical response,☆☆

https://doi.org/10.1016/S0016-5085(99)70405-1Get rights and content

Abstract

Background & Aims: Patients with chronic hepatitis C virus infection are commonly treated with interferon alfa (IFN-α), but the long-term response rate is poor. A region of NS5A of hepatitis C virus genotype 1 (the ISDR) has been associated with treatment outcome in some patients. NS5A binds to and inhibits PKR in vitro and inhibits IFN-α in human cells. We examined the effects of the NS5A protein from patients who did or did not respond to IFN-α to determine whether NS5A from IFN-α nonresponders inhibited the effects of IFN-α in vitro. Methods: We cloned NS5A from patients who had well-characterized responses to IFN-α and expressed them in a human fibroblast cell line under the control of an inducible promoter. The NS5A expression levels were controlled, and the effects of different proteins on the protective actions of IFN-α against encephalomyocarditis virus were investigated. Results: NS5A expression blocked the antiviral effects of IFN-α in human cells. This inhibition was dependent on the level of NS5A expression. Although ISDR changes gave only small differences in IFN-α inhibition, clones derived from a patient who did not respond to IFN-α and one who did respond to treatment differed greatly: the clones from a patient with response to IFN-α were much more inhibitory than those derived from the patient with no response. Conclusions: The inhibition of the antiviral effects of IFN-α by NS5A is not regulated exclusively by the ISDR, and the effects of NS5A in vitro do not correlate with treatment outcomes.

GASTROENTEROLOGY 1999;117:1187-1197

Section snippets

Patients

Full-length NS5A was amplified by reverse-transcription polymerase chain reaction (RT-PCR) from sera from the serum bank at St. Mary's Hospital (London, England). Patients had a diagnosis of isolated chronic HCV and had received 4.5 MIU of IFN-α (Roferon) 3 times weekly for at least 12 weeks. Patients who remained HCV PCR negative (Roche Amplicor assay; Sommerville, NJ) after 12 weeks were treated for an additional 9 months, and patients who were viremic (PCR positive) after 12 weeks had their

NS5A expression in HT1080 cells

Attempts to generate HuH-7 and HepG2 stable cell lines with constitutive NS5A expression failed. Using the Ecdysone-Inducible Expression Kit (Invitrogen) and HT1080 cells (a human fibroblastoid cell line), NS5A could be expressed. In this system, NS5A expression is induced by treating cells with muristerone A. [methyl-3H]Thymidine incorporation assays showed that although NS5A expression over 24 hours reduced the cell growth rate, short-lived expression was not toxic (data not shown). NS5A

Discussion

Patients with chronic HCV infection are commonly treated with IFN-α, but the long-term response rate is poor. Previous studies suggest that amino acid mutations in the ISDR region of HCV NS5A may be related to IFN treatment outcome.14, 16 Recent studies show that the NS5A interaction and inhibition of PKR in vitro is dependent on ISDR amino acid sequence22, 23: NS5A-1b clones modified to include ISDR motifs predicted to confer resistance to IFN therapy in patients inhibited PKR in vitro,

Acknowledgements

The authors thank the following for their generous reagent gifts: HT1080 cells from D. Watling (Imperial Cancer Research Fund, London, England); pooled rabbit anti-NS5A and anti-NS5B serum from F. Hoffmann–La Roche Ltd. (Basel, Switzerland); the NS5BGDD→PDD clone used to generate ecdysone-inducible cell line from G. Canning and H. Overton (Roche Discovery, Welwyn, England); and the 6–16 probe from S. Goodbourn (St. George's Hospital, London, England). The authors also thank Karen Gutekunst

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    Address requests for reprints to: Graham R. Foster, M.D., Department of Medicine, 10th Floor, QEQM Wing, Imperial College of Medicine at St. Mary's Campus, Norfolk Place, London, W2 1PG, England. e-mail: [email protected].

    ☆☆

    Supported in part by Medical Research Council grant G78/4839.

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