Hazari, S, Chandra, PK, Poat, B, Datta, S, Garry, RF, Foster, TP et al.. Impaired antiviral activity of interferon alpha against hepatitis C virus 2a in Huh-7 cells with a defective Jak-Stat pathway. Virol J 7: 36

Department of Pathology and Laboratory Medicine, Tulane University of Health Sciences Center, 1430 Tulane Ave, New Orleans, LA 70112, USA.
Virology Journal (Impact Factor: 2.18). 02/2010; 7(1):36. DOI: 10.1186/1743-422X-7-36
Source: PubMed


The sustained virological response to interferon-alpha (IFN-alpha) in individuals infected with hepatitis C virus (HCV) genotype 1 is only 50%, but is about 80% in patients infected with genotype 2-6 viruses. The molecular mechanisms explaining the differences in IFN-alpha responsiveness between HCV 1 and other genotypes have not been elucidated.
Virus and host cellular factors contributing to IFN responsiveness were analyzed using a green fluorescence protein (GFP) based replication system of HCV 2a and Huh-7 cell clones that either possesses or lack a functional Jak-Stat pathway. The GFP gene was inserted into the C-terminal non-structural protein 5A of HCV 2a full-length and sub-genomic clones. Both HCV clones replicated to a high level in Huh-7 cells and could be visualized by either fluorescence microscopy or flow cytometric analysis. Huh-7 cells transfected with the GFP tagged HCV 2a genome produced infectious virus particles and the replication of fluorescence virus particles was demonstrated in naïve Huh-7.5 cells after infection. IFN-alpha effectively inhibited the replication of full-length as well as sub-genomic HCV 2a clones in Huh-7 cells with a functional Jak-Stat pathway. However, the antiviral effect of IFN-alpha against HCV 2a virus was not observed in Huh-7 cell clones with a defect in Jak-Stat signaling. HCV infection or replication did not alter IFN-alpha induced Stat phosphorylation or ISRE promoter-luciferase activity in both the sensitive and resistant Huh-7 cell clones.
The cellular Jak-Stat pathway is critical for a successful IFN-alpha antiviral response against HCV 2a. HCV infection or replication did not alter signaling by the Jak-Stat pathway. GFP labeled JFH1 2a replicon based stable cell lines with IFN sensitive and IFN resistant phenotypes can be used to develop new strategies to overcome IFN-resistance against hepatitis C.

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Available from: Sibnarayan Datta
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    • "Another recent review described the modulation of HCV infection by JAK-STAT signaling via multiple mechanisms [15]. The importance of cellular JAK-STAT signaling in the IFN-α antiviral response has also been confirmed in our laboratory using stable sub-genomic HCV replicon cell lines, as well as an infectious HCV cell culture system [16]–[18]. We demonstrated that HCV replication in a replicon cell line expressing a truncated IFNAR1 remained resistant to IFN-α treatment. "
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    ABSTRACT: Purpose Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients. Methods Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting. Result HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rβ (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients. Conclusion HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "The effect of FFA treatment on HCV replication and IFNAR1 expression was measured by flow analysis as described previously [21]. Briefly, S3-GFP replicon cells with or without FFA treatment were collected 24 h post- treatment. "
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    ABSTRACT: Background Hepatic steatosis is recognized as a major risk factor for liver disease progression and impaired response to interferon based therapy in chronic hepatitis C (CHC) patients. The mechanism of response to interferon-alpha (IFN-α) therapy under the condition of hepatic steatosis is unexplored. We investigated the effect of hepatocellular steatosis on hepatitis C virus (HCV) replication and IFN-α antiviral response in a cell culture model. Methods Sub-genomic replicon (S3-GFP) and HCV infected Huh-7.5 cells were cultured with a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in these cells was visualized by Nile red staining and electron microscopy then quantified by microfluorometry. The effect of FFA treatment on HCV replication and IFN-α antiviral response was measured by flow cytometric analysis, Renilla luciferase activity, and real-time RT-PCR. Results FFA treatment induced dose dependent hepatocellular steatosis and lipid droplet accumulation in the HCV replicon cells was confirmed by Nile red staining, microfluorometry, and by electron microscopy. Intracellular fat accumulation supports replication more in the persistently HCV infected culture than in the sub-genomic replicon (S3-GFP) cell line. FFA treatment also partially blocked IFN-α response and viral clearance by reducing the phosphorylation of Stat1 and Stat2 dependent IFN-β promoter activation. We show that FFA treatment induces endoplasmic reticulum (ER) stress response and down regulates the IFNAR1 chain of the type I IFN receptor leading to defective Jak-Stat signaling and impaired antiviral response. Conclusion These results suggest that intracellular fat accumulation in HCV cell culture induces ER stress, defective Jak-Stat signaling, and attenuates the antiviral response, thus providing an explanation to the clinical observation regarding how hepatocellular steatosis influences IFN-α response in CHC.
    Full-text · Article · Aug 2012 · Virology Journal
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    • "The Huh-7 cells obtained from the laboratory of James Wilson, Wistar Institute of Human Gene Therapy, Pennsylvania and R4GFP HCV (+) replicon cells were previously developed in our laboratory (Hazari et al., 2010). The continuous replication of HCV in R4GFP HCV (+) replicon cells was monitored by GFP expression. "
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    ABSTRACT: The goal of this study is to evaluate the stability of lyophilized siRNA formulations. The gene silencing efficiency of a stored lyophilized siRNA formulation (i.e. siRNA nanosomes) was evaluated in interferon-α (IFN-α) resistant hepatitis C virus (HCV) at different time points up to three months in an in vitro cell culture model and compared with freshly prepared siRNA formulations. Novel siRNA sequences were encapsulated within nanosize liposomes following condensation with protamine sulfate. The siRNA encapsulated nanosomes were lyophilized and stored at 4 °C for 3 months, along with liquid liposomes (L) and lyophilized liposome powder (P) which were subsequently used to prepare siRNA nanosomes (L) and siRNA nanosomes (P), respectively at different time points. Physiochemical and biological properties of all three formulations were compared at different time points up to 3 months. The particle size of the stored siRNA nanosomes (642 ± 25 nm) was considerably larger initially in comparison with the liquid liposomes (134 ± 5 nm) and lyophilized liposomes (118 ± 3). However, the particle size gradually became smaller over time (413 ± 128 nm by the third month). The zeta potential of all three formulations was initially very high (> +40 mV), followed by a gradual decrease over time. The amount of siRNA in the stored siRNA nanosomes decreased ∼18 % during the 3 month storage period (1.16 ± 0.03 nmol initially on day 1 vs. 0.95 ± 0.04 nmol after 3 months). With respect to biological potency, all three formulations were significantly effective to knock-down HCV throughout the storage time. The cell viability was well-maintained throughout this period. Thus, this study indicates that the stored lyophilized siRNA formulation is as effective as the fresh preparation and that long-term storage could be a viable option to treat deadly diseases such as cancer and viral infection.
    Full-text · Article · Dec 2011 · International Journal of Pharmaceutics
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