Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

Abstract

Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon-alpha/beta and antiviral/interferon-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome 3' non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo, and triggered interferon and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP-RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.

Figure 1. Identification of HCV PAMP RNA

a–d, RNA-induced IFN-β promoter-luciferase activity in Huh7 cells, shown as mean fold-index induction (compared to non-treated cells; ±SD). Huh7 cells were transfected with 1 µg (0.4 pmol ) of HCV N (HCV 1b) genome RNA, 1 µg of poly inosine:cytosine (pIC) RNA (control) or with 1 µg ( 5–10 pmol) of of the indicated RNA species and harvested for dual luciferase assay 16 h later. HCV 1b refers to HCV genome RNA; tRNA, transfer RNA control; b–d, nt numbers encoded by HCV RNA constructs are shown. Bars are placed in their relative positions of each region within the HCV genome shown in b. The 5’ NTR, protein coding regions, and 3’ NTR are indicated. e, The HCV 3’ NTR motifs and respective RNA constructs. RI and broken lines denote replication intermediate. f, IFN-β promoter activation, shown here and in remaining figures as mean relative luciferase units (RLU; ±SD), triggered by 1 µg (20-150 pmole) of the indicated RNA species in transfected Huh7 cells. g, The abundance of IRF-3, ISG56 and tubulin (control) were measured by immunoblot. The upper panel shows the active IRF-3 dimer and inactive monomer forms separated by nondenaturing PAGE. h, RNA binding/gel-shift analysis of purified RIG-I with poly-U/UC or X region RNA (6 pmol) reacted with 0, 10, 20, 40, or 60 pmol of RIG-I protein. All RNAs contain 5’ppp. Asterisks indicate significant difference (P<0.01) as determined by Student’s T-test.

Figure 2. RIG-I-specific HCV RNA PAMP recognition and signaling

a–e, Induction of the IFN-β promoter in cells co-transfected with 1 µg (20-30pmol) of tRNA control or the indicated HCV RNA species. a, Huh7.5 cells, lacking functional RIG-I, were cotransfected with a plasmid encoding vector alone or RIG-I. b, Promoter signaling in wild-type (wt) and RIG-I^−/−, c, MDA5^−/−, d, MyD88^−/− or e, TRIF^−/− mouse embryo fibroblasts. Asterisk indicate a significant difference (P<0.01) from tRNA control. f, FRET analysis of Cy3-labeled poly-U/UC RNA (Cy3-PUC) interaction with YFP-RIG-I or YFP-DAI protein in co-transfected Huh7 cells. Panels show representative images of YFP, Cy3, merged fluorescence, and N FRET (corrected FRET). The color scale denotes N FRET levels. The bar graph at right shows the calculated values for RNA interaction with RIG-I or DAI. Control values are from the image area that has no colocalization signal. All RNAs contain 5’ppp.

Figure 3. Poly-uridine and poly-adenosine ribonucleotides are RIG-I ligands

a, Gel shift analysis of complex formation between 25 pmol of purified N-RIG (control) or full-length RIG-I (FL) and 10 pmol of poly-U/UC (PU/UC) or X region RNA containing 5’ppp or 5’OH as indicated. Arrows denote position of unbound RNA and RNA/RIG-I complexes. b, Effect of 5’ppp on IFN-β promoter activity. Huh7 cells were either mock-treated or treated with IFN-β 8 h prior to transfection with 1 µg (30 pmol ) of RNA. c, Effect of poly-U/UC or X region RNA on RIG-I activation. The silver-stained gel image shows trypsin-digestion products of RIG-I that was pre-incubated with increasing amounts poly-U/UC or X region RNA. Arrows indicate positions of full length (FL) RIG-I and the 17 kDa trypsin-resistant RD of from RIG-I/RNA complexes. d, Effect of nt length of 1 µg (30-150 pmol) poly-U/UC 3’ truncation products on IFN-β promoter signaling in Huh7 cells. e–g, Effect of nt composition on IFN-β promoter signaling in Huh7 cells transfected with 1 µg (30 pmol ) of RNA. h, Effect of nt composition on RIG-I activation. The silver-stained gel image shows trypsin-digestion products of RIG-I that was pre-incubated with increasing amounts poly-U/UG, poly-C, poly-A, or poly-G RNA. Arrows indicate positions of full length RIG-I and the 17 kDa trypsin-resistant RD. We confirmed the 17 kDa fragment as the RIG-I RD by immunoblot analysis of the digestion products using an antiserum specific to the RIG-I carboxyl terminus (not shown), as previously described . Asterisks indicate significant difference (P<0.01) as determined by Student’s T-test.

Figure 4. HCV PAMP RNA triggers the hepatic innate immune response and anti-HCV defenses

a–f, Wild-type or RIG-I^−/− mice (n = 3) were hyrdrodynamically transfected intravenously with HCV RNA. a, mice received 100 μg of HCV 1b genome, HCV 1b genome lacking the 3’NTR (HCV 1b Δ 3’NTR), PU/UC RNA or X region RNA. Hepatic IFN-β mRNA expression was measured 8 hrs later. b–f, Wild-type or RIG-I ^−/− mice (n = 3) received 200 μg of poly-U/UC RNA or buffer control, and were sacrificed 4, 8 or 24 h later for comparative measurement of mRNA and protein expression. b, Liver-specific expression of IFN-β mRNA. c, Serum IFN-β protein levels. d, Liver-specific expression of RIG-I mRNA. e, Liver-specific expression of ISG56 mRNA. f, Immunohistochemcial stain of ISG56 protein expression in liver tissue sections. g and h, Paracrine antiviral effect of the innate immune response triggered by HCV PAMP RNA. g, Inhibition of HCV infection in pretreated cells. Triplicate cultures of Huh7.5 cells were treated with IFN-β or conditioned media collected from Huh7 cells transfected with the indicated RNA species for 12 h prior to HCV infection. The graph shows the number of infected cells (±SD) as determined by focus forming unit (FFU) assay at 48 h postinfection. h, Huh7.5 cells were infected with HCV for 48 h and then were treated with increasing concentrations of IFN-β or the indicated conditioned media for an additional 48 h. Intracellular HCV RNA levels relative to GAPDH were determined and are plotted as mean HCV RNA index (±SD) relative to infected, untreated cells.