Basic Studies Interferons a, b, g each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation Srikanta Dash 1 , Ramesh Prabhu 1 , Sidhartha Hazari 1 , Frank Bastian 1 , Robert Garry 2 , Weiping Zou 3 , Salima Haque 1 , Virendra Joshi 3 , Fredric G. Regenstein 3 and Swan N. Thung 4 1 Departments of Pathology and Laboratory Medicine, 2 Microbiology and Immunology, 3 Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, 4 Department of Pathology, The Mount Sinai Medical Center, New York, NY, USA Dash S, Prabhu R, Hazari S, Bastian F, Garry R, Zou W, Haque S, Joshi V, Regenstein FG, Thung SN. Interferons a, b, g each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation. Liver International 2005: 25: 580–594. r Blackwell Munksgaard 2005 Abstract: Interferon (IFN)-a is the standard therapy for the treatment of chronic hepatitis C, but the mechanisms underlying its antiviral action are not well understood. In this report, we demonstrated that IFN-a,-b and -g inhibit replication of the hepatitis C virus (HCV) in a cell culture model at concentrations between 10 and 100 IU/ml. We demonstrated that the antiviral actions each of each these IFNs are targeted to the highly conserved 5 0 untranslated region of the HCV genome, and that they directly inhibit translation from a chimeric clone between full-length HCV genome and green fluorescent protein (GFP). This effect is not limited to HCV internal ribosome entry site (IRES), since these IFNs also inhibit translation of the encephalomyocardititis virus (EMCV) chimeric mRNA in which GFP is expressed by IRES-dependent mechanisms (pCITE-GFP). These IFNs had minimal effects on the expression of mRNAs from clones in which translation is not IRES dependent. We conclude that IFN-a,-b and -g inhibit replication of sub-genomic HCV RNA in a cell culture model by directly inhibiting two internal translation initiation sites of HCV- and EMCV-IRES sequences present in the dicistronic HCV sub-genomic RNA. Results of this in vitro study suggest that selective inhibition of IRES-mediated translation of viral polyprotein is a general mechanism by which IFNs inhibits HCV replication. Key words: hepatitis C virus – replicates – translation – IRES – T7 RNA polymerase – RPA Srikanta Dash, PhD, Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Ave., SL-79, New Orleans, LA 70112, USA. Tel: 504-988-2519 Fax: 504-988-7389 e-mail: sdash@tulane.edu Received 4 May 2004, accepted 13 September 2004                                                                                 Hepatitis C virus infection (HCV) is a global health problem with approximately 200 million people estimated to be infected worldwide. It is an enveloped positive-stranded RNA virus, belong- ing to the Flaviviridae family (1–3). Approxi- mately 75% of individuals after initial exposure to HCV develop chronic infection (4–6). Failure to mount a successful immune response and clear the virus results in the establishment of a chronic carrier state (7, 8). Of the patients chronically infected, up to 25% are at risk of developing cirrhosis and hepatocellular carcinoma. At pre- sent no effective immunoprophylaxis or candi- date vaccine is available against HCV. Interferon (IFN) a in combination with ribavirin, is the standard therapy for patients with hepatitis C (9–12). However, in most patients the sustained response rate is only 50%, even with the newest therapeutic regimens (13). The reasons why nearly half of HCV-treated patients fail to re- spond to standard IFN therapy are unknown. IFNs are the super family of proteins secreted by human cells that manifest multiple functions in the human body. They serve a role in protect- ing cells from viral infection, regulating cell growth and modulating the immune system (14– 19). Different IFNs vary in binding to their receptors; IFN-a and IFN-b bind to IFN-R1, whereas IFN-g binds to IFN-R2. There is con- siderable overlap in the signal transduction path- ways between the different IFNs (20). The biological activities of IFNs are initiated by the binding to these receptors on the surface of cells, which results in the activation of two receptor- associated protein tyrosine kinases, Jak1 and Tyk2 (21–23). Phosphorylation of the IFN recep- tor subunits by the activated JAK provides re- cruitment, phosphorylation and dimerization of signaling molecules that are members of the signal transducer and activator of transcription (Stats) family. The cascades of biochemical reac- tion occurring in normal cells because of IFN Liver International 2005: 25: 580–594 Printed in Denmark. All rights reserved Copyright r Blackwell Munksgaard 2005 DOI: 10.1111/j.1478-3231.2005.01082.x 580