Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner

Abstract

The impact of hepatitis C virus NS5A protein mutations on interferon alfa (IFN-␣) signaling pathway, cell proliferation, and viability is an important issue that is still under debate. We have therefore combined transient and stable expression in a human hepatocytic cell line (Huh7) of 3 full-length NS5A sequences, isolated from patients with or without response to IFN-␣ therapy. Expression of all 3 NS5A-reduced IFN-␣ global antiviral activity on both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) replication. We did not show, however, an effect of these 3 NS5A proteins on double-stranded RNA-dependent kinase (PKR) expression and activity as well as colocalization and coimmunoprecipitation between NS5A and PKR. We also failed to show an effect of the 3 NS5A mutants tested on cell proliferation and viability. Overall Hepatitis C virus (HCV) is a positive-stranded RNA virus classified as hepacivirus in the Flaviridae family, which exhibits marked viral heterogeneity. 1 The polyprotein precursor is co-and post-translationally processed by both cellular and viral proteases to yield mature, structural and nonstructural proteins. 2-4 HCV is a highly prevalent pathogen and at least 300 million individuals are chronically infected worldwide. 5 The establishment of chronic infection is a major feature of HCV infection, because around 70% to 80% of acutely infected subjects will become chronic carriers, with a high subsequent risk of progression to cirrhosis and hepatocarcinoma. 6 Therapy using interferon alfa (IFN-␣) alone, or in combination with ribavirin, has been shown to be effective in chronic hepatitis C infection, but only 8% to 12% and 30% to 40% of treated patients, respectively, depending on HCV type, viral load, and quasispecies complexity, show a sustained virologic response. [7][8][9] In this context, appraisal of the mechanisms of viral resistance to IFN-␣ therapy is crucial.Several studies have suggested a direct interplay between HCV and IFN-␣-induced cellular signaling. 10,11 Expression of HCV polyprotein in osteosarcoma cells inhibits Jak1-STAT signaling. 12 HCV nonstructural protein NS5A of HCV type 1 has been particularly investigated. In Japanese HCV-1b isolates, an inverse correlation was indeed observed between the number of mutations in a region of NS5A, referred to as the interferon-sensitivity determining region (ISDR; position 2209-2248), and the response to IFN-␣ treatment. 13,14 Such a strict correlation between ISDR mutations and treatment efficacy has not, however, been shown in most studies performed in Europe and the United States. [15][16][17][18][19][20][21] Furthermore, NS5A sequence variability in domains located outside the ISDR, and in particular in the C-terminal part of the protein, is probably also implicated in therapeutic efficacy. 22 Expression of fulllength NS5A-1b but not of ISDR-deleted mutant, renders osteosarcoma 11 and murin fibroblastic cell lines 23 partially resistant to the antiviral effect of IFN-␣ against encephalomyocarditis virus (EMCV) an...

Activation of the interferon-inducible protein kinase PKR by Hepatocellular carcinoma derived-Hepatitis C virus core protein

“…Some studies have suggested that NS5A may bind PKR and inhibits its activity (Gale et al, 1998). We recently reported, however, that at least in Huh7 cells, we did not show such an interaction (Podevin et al, 2001). These very important results turned to advantage, providing additional evidence for the speci®city of our present observations on HCV core.…”

“…HCV polyprotein expression impairs interferon-dependent signal transduction and in particular STAT DNA binding e cacy (Gale et al, 1998;Heim et al, 1999;Polyak et al, 1999). Furthermore, it has been proposed that two HCV proteins: E2 and NS5A, may bind PKR, inhibit its antiviral and anti-apoptotic activities and thus favor HCV chronicity and liver cell clone expansion (Gale et al, 1998;Taylor et al, 1999); the results concerning NS5A may di er depending on the cell line examined and we were unable to show such an association in HuH7 cells (Podevin et al, 2001). Nevertheless, the present results allow us to propose an alternative, and in fact contrasting mechanism for HCV core protein.…”

Hepatitis C viral load does not predict disease outcome: going beyond numbers

“…Recently, PODEVIN et al 28 have extended the discussion about the role of NS5A, questioning previous models that did not analyze the role of NS5A in hepatocytes and whose results should therefore be considered with caution. In their study, the first one on hepatocytes, the authors analyzed human hepatocytic cell lines (Huh 7) expressing NS5A sequences derived from one responder and two non-responders in the presence of interferon-alpha and exposed to virus strains sensitive to the drug.…”

Quasispecies evolution in NS5A region of hepatitis C virus genotype 1b during interferon or combined interferon-ribavirin therapy

“…Recently, it was suggested that NS5A protein may also inhibit the antiviral effect of IFN in a PKR-independent manner in a human hepatocytic cell line [25] . Furthermore, a n o t h e r d o m a i n i n t h e N S 5 A r e g i o n , n a m e d V 3 (NS5A2356-2379) [26] , could also be linked to IFN resistance.…”