HCV F/ARFP suppresses ISGs, pro-inflammatory cytokines, and type III IFN responses in Huh7 cells.

<p>(A—B) Huh7 cells were transfected with indicated plasmids and, after 48 hrs, transfected with 5 μg of HCV RNA PAMP corresponding to the UTR or poly(IC). Samples were analyzed for the indicated mRNAs 24 hrs after stimulation with PAMP by qRT-PCR. Data were normalized by GAPDH mRNA and shown as percentage of controls. Star indicates statistically significant difference (P < 0.05) from respective minus PAMP controls. Letter “a” indicates statistically significant difference (P < 0.05) from the corresponding pEF controls for each -PAMP or +PAMP group. Lines with P values also indicate statistically significant difference between those samples.</p

Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I

<div><p>Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of <u>f</u>rameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back <i>via trans</i>-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity.</p></div

Role of RIG-I in the modulation of IFNβ1 by HCV -2/+1 frame mutants.

<p>(A) Huh7 and Huh7.5 cells were transfected with JFH1wt, JFH1Δ, and JFH1Δ4 RNAs or mock-transfected and analyzed for IFNβ1 mRNA and HCV RNA by qRT-PCR after 72 hrs. (B) Huh7 cells were transfected with siRNAs and then transfected with JFH1wt and JFH1Δ4 RNAs or mock-transfected 48–72 hrs later. Cells were then analyzed for IFNβ1 and RIG-I mRNAs by qRT-PCR. Data were normalized by GAPDH mRNA. (C) Huh7 cells supporting JFH1wt, JFH1Δ, and JFH1Δ4 were incubated with 0, 100, or 250 U/ml of exogenous IFNα (NIAID Reference Reagent Repository and Sigma Aldrich) with change of cell culture medium daily for 72 hrs. Then, samples were collected and analyzed by qRT-PCR. Data are shown as percentage of respective 0 U/ml IFNα controls. Star indicates statistically significant difference (P < 0.05) compared to controls. Lines with P values also indicate statistically significant difference between those samples.</p

List of qRT-PCR primers.

<p>List of qRT-PCR primers.</p

Effects of HCV core versus F/ARFP on IFNβ1 mRNA elevation by JFH1Δ4.

<p>(A) Huh7 cells were transfected with pCoreΔ or pF as well as JFH1wt or JFH1Δ4 RNA and analyzed for IFNβ1 mRNA by qRT-PCR. Data are normalized by JFH1 RNA and GAPDH mRNA levels and expressed as percentage of pCoreΔ/JFH1wt transfected control. Star indicates statistically significant difference (P < 0.05) from JFH1wt for each plasmid group; letter “a” indicates statistically significant difference (P < 0.05) from pCoreΔ. (B) Huh7 cells transfected with pCoreΔ4, pHA-F, pF, and pFLAG-NS3/4A were analyzed for IFNβ1 mRNA by qRT-PCR. Data were normalized by GAPDH mRNA. Star indicates statistically significant difference (P < 0.05) from respective minus PAMP controls. Letter “a” indicates statistically significant difference (P value less than 0.05) from pCoreΔ4.</p

Schematics of HCV -2/+1 frame mutants.

<p>(A) Putative -2/+1 frame protein products of HCV. Translational frameshift sites are indicated with bent arrows. White bars represent zero frame and gray bars, protein regions coded by the -2/+1 frame. Dotted lines indicate positions where the majority of -2/+1 frame sequences terminate, such as stop codon at codon 126 in the -2/+1 frame for JFH1. Numbers represent codons, and locations of Δ and Δ4 mutations are marked with stars. (B) JFH1 constructs. Putative RNA elements for the generation of various -2/+1 elements and nt. substitutions introduced in JFH1 constructs are shown. Numbers represent nucleotide positions within the JFH1 polyprotein sequence.</p

F/ARFP detection in human liver samples and concentration-dependent suppression of IFN response by F/ARFP.

<p>(A) Huh7 cells transfected with pEF, pCoreΔ4, or pFΔ (2 μg each) and HCV-infected and HCV–uninfected human liver samples (n = 3 for infected, n = 2 for uninfected) were analyzed for HCV core and F/ARFP proteins by immunofluorescence analysis using corresponding antibodies. Scale bar (20 μm) is the same for all the pictures, but is only shown in one of each experiment. (B) Immunofluorescence images were analyzed by ImageJ and shown as change in average intensities for HCV-uninfected and–infected liver samples. (C) Huh7 cells (3 × 10⁵) were transfected with different amounts of pHA-F and pCoreΔ4 plasmids and, after 24 hrs, stimulated with poly(IC), and analyzed for IFNβ1 mRNA after another 24 hrs. Star indicates statistically significant difference (P < 0.05) from the control group. Letter “a” indicates statistically significant difference (P < 0.05) from the corresponding pEF control for each–poly(IC) or +poly(IC) group. Lines with P values also indicate statistically significance (P < 0.05) or no difference (P > 0.05) between the groups. Data were normalized by GAPDH mRNA and shown as percent change from control.</p

Suppression of type I IFN induction by HCV F/ARFP.

<p>(A) Huh7 cells transfected with pEF (empty vector), pHA-F, pFLAG-NS3/4A, or pHA-F plus pFLAG-NS3/4A were analyzed by Western blots using anti-HA, FLAG, and actin antibodies. (B—D) Huh7 cells were transfected with indicated plasmids and, after 48 hrs, transfected with 5 μg of HCV RNA PAMP corresponding to the UTR or poly(IC). Samples were analyzed for the indicated mRNAs, 24 hrs after PAMP stimulation by qRT-PCR. For (B–D), star indicates statistically significant difference (P < 0.05) from respective minus PAMP controls. Letter “a” indicates statistically significant difference (P < 0.05) from the corresponding pEF control for each -PAMP or +PAMP group. Lines with P values also indicate statistically significant difference between those samples. All mRNA data were normalized by GAPDH mRNA and shown as percentage of controls.</p

Frameshift signal sequences in frameshift reporter constructs.

<p>Frameshift signal sequences in frameshift reporter constructs.</p

Induction of type I IFNs by HCV -2/+1 frame mutants.

<p>(A, B) Huh7 cells transfected with JFH1wt, JFH1Δ, JFH1Δ4, or no RNA (mock transfection control) were analyzed for IFNβ1, IFNα8, and RIG-I mRNAs or HCV RNA by qRT-PCR. (C) Cell culture media which were not concentrated were analyzed for secreted IFNβ1 by ELISA. (D) Medium harvested from the indicated viral RNA-transfected Huh7 cells were used to infect naïve Huh7 cells. Cells were then analyzed for IFNβ1 mRNA by qRT-PCR. qRT-PCR data were normalized by GAPDH mRNA and expressed as percentage of controls. Star indicates statistically significant difference (P < 0.05) from controls.</p