Liver, Pancreas, and Biliary TractHepatitis C virus NS5A protein inhibits interferon antiviral activity, but the effects do not correlate with clinical response☆,☆☆
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
References (30)
- et al.
Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome
Biochem Biophys Res Commun
(1994) - et al.
Viral inhibition of the interferon system
Pharmacol Ther
(1992) - et al.
Mutations in the nonstructural 5A region of hepatitis C virus and response of chronic hepatitis C to interferon alfa
Gastroenterology
(1997) - et al.
Mutations of hepatitis C virus 1b NS5A 2209–2248 amino acid sequence do not predict the response to recombinant interferon-alfa therapy in French patients
J Hepatol
(1997) - et al.
Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein
Virology
(1997) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)- et al.
Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis
N Engl J Med
(1989) Global surveillance and control of HCV
J Viral Hepat
(1999)- et al.
Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome
Science
(1989) - et al.
Genetic organisation and diversity of the hepatitis C virus
Proc Natl Acad Sci USA
(1991)
Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome
J Gen Virol
Expression and identification of hepatitis C virus polyprotein cleavage products
J Virol
Phosphorylation of hepatitis C virus–encoded nonstructural protein NS5A
J Virol
The N-terminal region of hepatitis C virus–encoded NS5A is important for NS4A-dependent phosphorylation
J Virol
Long-term follow-up of patients with chronic hepatitis C treated with alpha-interferon
Hepatology
Cited by (0)
- ☆
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.