Mechanism of HCV's resistance to IFN-α in cell culture involves expression of functional IFN-α receptor 1

Hauser Hansjörg, Datta Sibnarayan, Hazari Sidhartha, Chandra, Samara¹ Maria, Poat Bret, Gunduz Feyza, Wimley, Koster Mario, Lamaze Christophe, Balart, Garry, Dash Srikanta

Description

The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE- luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.

Sujets

• Unfolded protein response

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Dattaet al.Virology Journal2011,8:351 http://www.virologyj.com/content/8/1/351

R E S E A R C HOpen Access Mechanism of HCV’s resistance to IFNain cell culture involves expression of functional IFNa receptor 1 1†1†1†41 1 Sibnarayan Datta, Sidhartha Hazari, Partha K Chandra, Maria Samara¹ , Bret Poat , Feyza Gunduz , 3 55 64 2 William C Wimley , Hansjorg Hauser , Mario Koster , Christophe Lamaze , Luis A Balart , Robert F Garryand 1,4* Srikanta Dash

Abstract The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-a) are not fully understood. We used IFN-aresistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-aresistance in cell culture. The IFN-aresistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-areceptor 1 (IFNAR1), IFN-areceptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE- luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-aresistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-aresistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-a. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-a. Keywords:Hepatitis C virus (HCV), Interferon alpha (IFN-α), Interferon alpha-receptor 1 (IFNAR1), Jak-Stat signaling, nuclear translocation, reverse transcription polymerase chain reaction (RT-PCR), HCV infection, ER stress

Introduction Hepatitis C virus (HCV) is a positivestranded RNA virus that infects the liver. The majority of patients after initial exposure to the virus develop a chronic infection. Chronic HCV infection can gradually evolve into liver cirrhosis, end stage liver diseases and hepatocellular car cinoma [13]. The standard treatment option of chronic HCV infection is the combination of IFNaand riba virin [4]. This therapy cures approximately 50% of

* Correspondence: [e-mail] †Contributed equally 1 Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, USA Full list of author information is available at the end of the article

chronic HCV infections and the HCV in a majority of chronically infected patients develop resistance. The mechanism of IFNaresistance in these patient popula tions is not fully understood. Understanding the IFNa resistance mechanism of HCV infection is important to develop an alternative therapeutic strategy to clear the infection. To understand the mechanism of HCV resistance to IFNa, we have utilized stable replicon cell lines and the infectious HCV cell culture model system. The replicon cells express NS3 to NS5B protein required for replica tion of HCV subgenomic RNA but they lack structural proteins and do not produce infectious virus. We have isolated nine stable IFNaresistant Huh7 based

© 2011 Datta et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.