Hepatitis C virus NS5A protein modulates IRF-7-mediated interferon-α signaling

Abstract

Hepatitis C virus (HCV) establishes chronic infection in a large number of infected individuals. We have previously shown that HCV infection in hepatocytes blocks poly (I-C) or interferon (IFN)-α-mediated IRF-7 nuclear translocation (Raychoudhuri and others 2010). However, the mechanism of IRF-7 regulation by HCV remained unknown. In this study, we have observed that HCV NS5A physically associates with IRF-7. A subsequent study suggested that the HCV NS5A protein blocks IRF-7-mediated IFN-α14 promoter activation. Further analyses demonstrated that site-specific mutagenesis of the 2 basic arginine residues (amino acids Arg(216) and Arg(217)) in the NS5A is critical for IRF-7-mediated IFN-α14 promoter regulation. Together, our results suggested that the HCV NS5A protein limits the IFN-α-signaling pathway in association with IRF-7, and may, in part, be responsible for the establishment of chronic infection.

FIG. 1.

Hepatitis C virus (HCV) NS5A suppresses IRF-7-mediated interferon (IFN)-α14 promoter activity. (A) Immortalized human hepatocytes (IHH) were cotransfected with human IFN-α4, IFN-α7, or IFN-α14 promoter with the Luc reporter and IRF-7 plasmid constructs. Cell extracts were prepared 48 h post-transfection and the luciferase activity was measured. (B) IHH were cotransfected with human IFN-α14-luc reporter plasmid, IRF-7, and HCV genomic region encoding specific protein(s). Cell extracts were prepared for luciferase assay 48 h post-transfection. (C) IHH were cotransfected with human IFN-α14-luc reporter plasmid, IRF-7 construct, and/or HCV NS5A. Cell extracts were prepared for luciferase assay 48 h post-transfection. Results presented are mean with standard error from 3 independent experiments. Empty vector DNA was used as a negative control and the mean basal value was arbitrarily set at 1 for all luciferase assays.

FIG. 2.

HCV NS5A associates with IRF-7. (A, B) IRF-7-GFP or control vector was transfected into Huh-7 cells harboring HCV subgenomic replicon from genotype 1b (A) or genotype 2a (B). Cell lysates were prepared 48 h post-transfection, and immunoprecipitated with the anti-GFP antibody. Association of IRF-7 and HCV NS5A was detected by immunoblotting with specific antibodies. The IRF-7 protein was detected with a specific antibody. (C) Human embryonic kidney 293 cells were cotransfected with IRF-7-GFP and control vector or HCV NS5A. Cell lysates were prepared after 48 h of transfection and immunoprecipitated with the anti-GFP antibody. A specific antibody was used for detection of HCV NS5A or IRF-7.

FIG. 3.

NS5A Arg²¹⁶ and Arg²¹⁷ are critical for inhibition of IRF-7-mediated IFN-α14 promoter activation. (A) Alignment of partial NS5A sequences from amino acid residues 190–269 among different genotypes is shown. The sequence analyses displayed conserved arginine residues marked in yellow. (B) IHH were cotransfected with human IFN-α14-luc reporter plasmid, IRF-7, and wild-type or mutant HCV NS5A constructs. The luciferase activity was measured 48 h post-transfection. Results presented are mean with standard error from 4 independent experiments. Empty vector DNA was used as a negative control and mean basal value was arbitrarily set at 1. (C) HCV NS5A (wild-type or mutant) protein expression is shown by immunoblotting with the FLAG antibody for detection of the NS5A protein. (D) IHH were cotransfected with IRF-7-GFP and wild-type or mutant HCV NS5A. Cells were stained after 48 h of transfection for expression of NS5A (red) and IRF-7 (green). Cell nuclei were stained with DAPI (blue). Cytoplasmic localization of IRF-7 and NS5A was observed from images superimposed digitally for fine comparisons in confocal microscopy. (E) IRF-7-GFP plasmid DNA was cotransfected with wild-type NS5A (Wt NS5A) or mutant NS5A (Mt NS5A) into 293 cells. Cell lysates were prepared after 48 h of transfection and immunoprecipitated with the anti-GFP antibody. Association of IRF-7 and wild-type or mutant NS5A was detected by immunoblotting with specific antibodies.