Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling

Abstract

Hepatitis C virus (HCV) is a major human pathogen that infects 170 million people. A hallmark of HCV is its ability to establish persistent infections reflecting the evasion of host immunity and interference with alpha/beta-IFN innate immune defenses. We demonstrate that disruption of retinoic acid-inducible gene I (RIG-I) signaling by the viral NS3/4A protease contributes to the ability of HCV to control innate antiviral defenses. RIG-I was essential for virus or HCV RNA-induced signaling to the IFN-beta promoter in human hepatoma cells. This signaling was disrupted by the protease activity of NS3/4A, which ablates RIG-I signaling of downstream IFN regulatory factor 3 and NF-kappaB activation, attenuating expression of host antiviral defense genes and interrupting an IFN amplification loop that otherwise suppresses HCV replication. Treatment of cells with an active site inhibitor of the NS3/4A protease relieved this suppression and restored intracellular antiviral defenses. Thus, NS3/4A control of RIG-I supports HCV persistence by preventing IFN regulatory factor 3 and NF-kappaB activation. Our results demonstrate that these processes are amenable to restoration through pharmacologic inhibition of viral protease function.

Fig. 1.

RIG-I and NS3/4A regulate innate intracellular defenses. (A) Structural representation of RIG-I, N-RIG, and C-RIG expression constructs. The positions of the caspase recruitment domain, helicase domain, and terminal amino acids are indicated. (B) Genome structure of HCV (Top). Also shown are the genome-length (Middle), and subgenomic HCV RNA replicons (Bottom) used in this study. These autonomously replicating, bicistronic viral RNAs express neomycin phosphotransferase (neo) and are thus selectable. They have been described in detail (6, 10). (C) Huh7 cells transfected with control siRNA or siRNA directed against TRIF or RIG-I were subsequently transfected with plasmids encoding the IFN-β luciferase reporter construct and a constitutively expressed β-galactosidase control construct, mock-infected or infected with SenV and harvested 20 h later for determination of relative luciferase values and RT-PCR analysis of mRNA expression. Bars show average and SD relative luciferase activity from three experiments. Panels at right show agarose gel analysis of the indicated RT-PCR product from cells treated as shown above each lane. The + and - RT designate control RT-PCR reactions to amplify glyceraldehyde dehydrogenase (GAPDH) mRNA in the presence or absence of reverse transcriptase. (D) Huh7 cells were cotransfected with plasmids encoding the IFN-β luciferase reporter construct, Renilla luciferase, and 50 ng of the indicated vector or RIG-I expression construct with increasing amounts (0, 50, 100, and 200 ng) of plasmid DNA encoding NS3/4A. Cells were mock-infected or infected with SenV and harvested for luciferase assay. Bars show the average relative luciferase activity and SD from three experiments. (E) Huh7.5 cells transfected with plasmid constructs encoding vector alone, RIG-I, or N-RIG in the presence of increasing amounts of an NS3/4A expression construct were infected with SenV as indicated and then were harvested for immunoblot analysis.

Fig. 2.

RIG-I signals IRF-3 to control HCV replication and is regulated by NS3/4A. (A and B) Control Huh7 cells were transfected with vector only. Huh7-A7 cells harboring the A7 HCV replicon (A7) or Huh7-HP cells harboring the HP HCV replicon (HP) were transfected with 1 μg of plasmid DNA encoding vector alone, IRF-3-5D or IRF-3-ΔNor1 μgor2 μg of N-RIG expression plasmid. Cells were harvested 48 h posttransfection, and extracts were subjected to Northern blot analysis (A) and immunoblot analysis (B) using specific DNA or antibody probes, respectively. (C) UNS3/4A cells, cultured to suppress (-NS3/4A) or induce (+NS3/4A) NS3/4A expression, were transfected with the N-RIG expression construct, and 24 h later were subjected to dual immunostaining for ectopic N-RIG and endogenous IRF-3. Panels show N-RIG, IRF-3, and DAPI-stained nuclei. (D) UNS3/4A cells cultured to suppress or induce NS3/4A expression were infected with NDV as shown (Left) or were transfected with N-RIG expression plasmid (Right). After 24 h, cells were harvested, and protein extracts were separated on nondenaturing gels and subjected to immunoblot analysis to define the dimer, monomer, and phosphoserine 386 (Ser-386-P) isoforms of IRF-3. (Bottom) NS3 levels derived by standard denaturing gel immunoblot analysis.

Fig. 3.

NS3/4A controls RIG-I signaling to NF-κB. (A and B) UNS3/4A cells, cultured to suppress (-NS3/4A) or induce (+NS3/4A) NS3/4A expression, were treated with IL-1, mock-infected or infected with SenV. (A) Cells were harvested at the times indicated, and extracts were subjected to immunoblot analysis to detect NS3, the phosphoserine 396 isoform of IRF-3 (IRF-3 Ser-396-P), total IRF-3, IκB-α, ISG56, and actin. (B) Cells were harvested 30 min post-IL-1 treatment or 16 h postinfection. Nuclear extracts were prepared and subjected to EMSA by using a DNA probe encoding the PRDII element of the IFN-β promoter to detect NF-κB DNA-binding activity. (C) Huh7 cells were cotransfected with plasmids encoding the PRDII-luciferase promoter construct and Renilla luciferase along with 50 ng of vector only or N-RIG expression plasmid and the indicated amount of NS3/4A expression plasmid. Cells were mock-infected or infected with SenV and processed for luciferase assay 16 h postinfection. Bars show the average relative luciferase and SD values from three experiments. (D) Huh7 2-3 cells harboring replicating genome-length RNA (HCV) or their IFN-cured Huh7 2-3c counterparts were cotransfected with plasmids encoding the PRDII-luciferase promoter construct and Renilla luciferase. Cells were mock-infected or infected with SenV and processed for luciferase assay. Bars show the average relative luciferase and SD values from three experiments. (E) Medium from cultures of mock or SenV-infected Huh7 2-3 (HCV) and Huh 2-3c cells (cured) was subjected to cytokine blot analysis by using the Cytokine Array III kit and the manufacturer's protocol (Ray Biotech, Norcross, GA). Numbers show the ratio of mock to SenV signal derived from averaged densitometric values of each spot. C denotes the reference control. (F) Huh7 2-3 (HCV) and Huh7 2-3c cells (cured) were mock-infected or infected with SenV for 16 h. Total RNA was extracted and subjected to microarray analysis by using Affymetrix U133A GeneChips. Bars show the quantified signal ratio of average hybridization levels for the indicated cytokine and chemokine mRNAs.

Fig. 4.

NS3/4A protease-dependent regulation of RIG-I disrupts an IFN amplification loop that controls HCV replication. (A and B) Transfected cells were cultured alone or in the presence of 10 μM SCH6 for 24 h before mock-infection or infection with SenV and luciferase assay. Bars show the average relative luciferase and SD values from three experiments. (A) Huh7 cells were cotransfected with plasmids encoding the IFN-β-luciferase promoter construct and Renilla luciferase along with 50 ng of plasmid DNA expressing vector only or RIG-I and 200 ng of plasmid DNA encoding WT NS3/4A, S1165A mutant NS3/4A, or IRF-3-ΔN as indicated. (B) UNS3/4A cells, cultured to suppress or induce NS3/4A expression as indicated, were cotransfected with plasmids encoding the PRDII-luciferase promoter construct and Renilla luciferase. (C) UNS3/4A cells, cultured to suppress or induce NS3/4A expression as indicated, were mock-infected, infected with SenV, or treated with 0, 10, 50, or 100 units/ml IFN-α2a as shown above each lane. Twenty hours later, cells were harvested for immunoblot analysis of endogenous protein levels. (D) Huh7 control or Huh7-A7, Huh7-K2040, and Huh-HP cells harboring the respective HCV replicon were mock-infected, infected with SenV, or treated with 50 units/ml IFN-α2a. After 20 h, the cells were harvested, and extracts were subjected to immunoblot analysis. (E) UNS3/4A cells, cultured to suppress or induce NS3/4A expression, were mock-infected, infected with SenV, or treated with 50 units/ml IFN-α2a. Twenty hours later, cells were harvested. Total RNA was isolated and reverse transcribed by using an oligo(dT) primer. Equal amounts of cDNA from each sample were subjected to quantitative real-time PCR analysis of RIG-I and GAPDH mRNA. Bars show the RIG-I mRNA level relative to the GAPDH internal control. (F) Huh7 cells (Left) or Huh7-HP cells harboring the HP HCV replicon (Right) were cultured in the presence of 10 μM SCH6 for the time indicated. Cells were harvested, and extracts were subjected to immunoblot analysis for determination of total IRF-3, the phosphoserine 396 isoform of IRF-3 (Ser-396-P), ISG56, NS5A, and actin levels. A parallel culture of Huh7 cells was infected with SenV and harvested 20 h later for use as an internal control (far left lane).