Direct, interferon-independent activation of the CXCL10 promoter by NF-κB and interferon regulatory factor 3 during hepatitis C virus infection

Abstract

Hepatitis C virus (HCV) infection of hepatocytes leads to transcriptional induction of the chemokine CXCL10, which is considered an interferon (IFN)-stimulated gene. However, we have recently shown that IFNs are not required for CXCL10 induction in hepatocytes during acute HCV infection. Since the CXCL10 promoter contains binding sites for several proinflammatory transcription factors, we investigated the contribution of these factors to CXCL10 transcriptional induction during HCV infection in vitro. Wild-type and mutant CXCL10 promoter-luciferase reporter constructs were used to identify critical sites of transcriptional regulation. The proximal IFN-stimulated response element (ISRE) and NF-κB binding sites positively regulated CXCL10 transcription during HCV infection as well as following exposure to poly(I·C) (a Toll-like receptor 3 [TLR3] stimulus) and 5' poly(U) HCV RNA (a retinoic acid-inducible gene I [RIG-I] stimulus) from two viral genotypes. Conversely, binding sites for AP-1 and CCAAT/enhancer-binding protein β (C/EBP-β) negatively regulated CXCL10 induction in response to TLR3 and RIG-I stimuli, while only C/EBP-β negatively regulated CXCL10 during HCV infection. We also demonstrated that interferon-regulatory factor 3 (IRF3) is transiently recruited to the proximal ISRE during HCV infection and localizes to the nucleus in HCV-infected primary human hepatocytes. Furthermore, IRF3 activated the CXCL10 promoter independently of type I or type III IFN signaling. The data indicate that sensing of HCV infection by RIG-I and TLR3 leads to direct recruitment of NF-κB and IRF3 to the CXCL10 promoter. Our study expands upon current knowledge regarding the mechanisms of CXCL10 induction in hepatocytes and lays the foundation for additional mechanistic studies that further elucidate the combinatorial and synergistic aspects of immune signaling pathways.

FIG 1

Schematic of the CXCL10 promoter-luciferase reporter constructs. Putative binding sites for NF-κB, AP-1, C/EBP-β, and the ISRE are labeled.

FIG 2

NF-κB and the proximal ISRE positively regulate the CXCL10 promoter in response to TLR3 and RIG-I PAMPs. Deletion of the NF-κB (κB1, κB2) (A) or proximal ISRE (B) binding sites eliminates activation of the CXCL10 promoter following 24 h of treatment with either exogenously added poly(I·C) (pIC; 5 μg/ml) or 0.5 μg transfected 5′ pU HCV PAMP (P < 0.05). Deletion of the AP-1 (C) or C/EBP-β (C/EBP-β1, C/EBP-β2) (D) binding site results in increased activity of the CXCL10 promoter in response to these stimuli (P < 0.01). Combination treatment with 100 ng/ml IFN-γ and 40 ng/ml TNF-α for 24 h served as a positive control for induction. Results are expressed as the mean ± standard deviation and are representative of two independent experimental repeats of three sample replicates each. RLU, relative light units. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

NF-κB and the proximal ISRE positively regulate the CXCL10 promoter in response to HCV infection. Deletion of the NF-κB (κB1, κB2) (A) or proximal ISRE (B) binding sites significantly decreased activation of the CXCL10 promoter following 24 h of HCV infection (P < 0.05; MOI, 1.0). (C) Deletion of the AP-1 binding site had no significant effect on the CXCL10 promoter response during HCV infection (P > 0.1). (D) Deletion of the C/EBP-β binding sites (C/EBP-β1, C/EBP-β2) increased the CXCL10 promoter response during HCV infection (P < 0.05). Results are expressed as the mean ± standard deviation and are representative of two independent experimental replicates of three sample replicates each. *, P < 0.05; **, P < 0.01.

FIG 4

HCV infection of PHHs activates IRF3 and the antiviral response. (A) Light microscope images of PHHs; (B) intracellular HCV RNA levels after infection with HCV (MOI, 5.0) for 12, 24, 36, and 48 h; (C) immunofluorescence analysis of IRF3 (red) and ISG15 (green) after infection with HCV (MOI, 5.0) for 24 h; (D) ISG15 and CXCL10 mRNA levels 36 h after infection with HCV (MOI, 5.0); (E) CXCL10 protein levels 36 h after infection with HCV (MOI, 5.0). Results are representative of staining performed with PHHs from two different donors.

FIG 5

Constitutively active IRF3 (IRF3-5D) drives CXCL10 transcription independently of type I and type III IFNs. Neutralization of type I IFNs via B18R (A) or type III IFNs via anti-IFN-λ (B) did not impact IRF3-5D-induced wild-type (WT) CXCL10 promoter activity 24 h after cotransfection in PH5CH8 immortalized hepatocytes. IRF3-5D was not able to induce expression of the ΔISRE CXCL10 mutant promoter above background levels induced by a control vector (pcDNA3.1 [pcDNA]). IFN-β promoter responses to IRF3-5D were included as a positive control. Results are expressed as the mean ± standard deviation and are representative of two independent experimental infections of three sample replicates each. #, conditions where addition of B18R or anti-IFN-λ led to a significant change in signal compared to that for nonneutralized samples (P < 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

IRF3 is recruited to the CXCL10 promoter during HCV infection. (A) Representative gel image of PCR products resulting from chromatin immunoprecipitation of TLR3⁺/RIG-I⁺ Huh7 cells infected with HCV (12 and 18 h; MOI, 0.6) or SeV (6 h; 100 HAU) using polyclonal anti-IRF3 serum. Normal rabbit serum (Rb Sera) was used as a negative control. Isolated chromatin fragments were PCR amplified using primers specific for the proximal ISRE of the CXCL10 promoter and resolved on an agarose gel. Input DNA was used as a positive control during PCR. Increased IRF3 binding to the CXCL10 promoter was detected during SeV infection and after 12 h of HCV infection. IRF3 binding returned to baseline levels 18 h after HCV infection. (B) Quantification of CXCL10 ISRE PCR band intensities expressed as a percentage of input DNA. Data were averaged from the results of three independent chromatin immunoprecipitation preparations. **, P < 0.01.