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

Abstract

Innate immune defenses 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) 1. Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people 2. Infection is governed by hepatic immune defenses triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals IRF-3 activation to induce the expression of α/β interferon (IFN) and antiviral/interferon-stimulated genes (ISGs) that limit infection 3 -10. Here we identified the poly-uridine motif of the HCV genome 3' nontranslated region (NTR) as the PAMP substrate of RIG-I, and show that this and similar homopoly-uridine motifs present in the genome of RNA viruses is the chief feature of RIG-I recognition and immune triggering 8. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent upon homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signaling to induce a hepatic innate immune response in vivo, and triggered IFN and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by identifying homopoly-uridine motfis present in the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and define immunogenic features of the PAMP/RIG-I interaction that could be utilized as an immune adjuvant for vaccine and immunotherapy approaches.To determine the nature of the HCV PAMP RNA we conducted a functional screen to identify possible HCV PAMP RNA motifs. We assessed the ability of full length HCV genome RNA or contiguous HCV RNA segments to trigger the IFN-β promoter in transfected Huh7 cells. The full-length HCV genome RNA triggered innate immune signaling to induce the IFN-β promoter (Fig. 1a). Two regions of the HCV RNA, encoding nt 2406-3256 and nt 8872-9616, stimulated significant induction of the IFN-β promoter (Fig. 1b) with signaling activity respectively localized to nt 2406-2696 of the open reading frame and nt 9389-9619 encoding the 3' NTR (Fig.1c). Deletion of nt 9389-9619 but not nt 2408-2663 from the HCV genome significantly attenuated signaling to the IFN-β promoter (Fig 1d). PAMP motifs are typically conserved among strains of a pathogen 1 , and sequence comparison of multiple HCV genomes revealed global variability within nt 2406-3696 among virus strains but nt 9389-9616 encoded motifs of high conservation (Fig. S1) Thus, the viral 3' NTR might encode HCV PAMP motifs that trigger innate immune signaling in the host cell.The HCV 3' NTR is comprised of three regions: a variable region (VR) with potential secondary structure, a nonstructured poly-U/UC region containing polyuridine with interspersed ribocytidine, and the terminal X region containing three conserved stem-loop structures (Fig. 1e) 12 . We evaluated the ability of RNA encoding the HCV 3' NTR or each of its regions to trigger intracellular si...

Dendritic Cell Inhibition Is Connected to Exhaustion of CD8+ T Cell Polyfunctionality during Chronic Hepatitis C Virus Infection

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Hepatitis C virus core protein triggers expansion and activation of CD4+CD25+ regulatory T cells in chronic hepatitis C patients

“…In the current study, we confirm that HCVc showed a significant, positive correlation with HCV RNA and the frequency of expression by blocking RIG-I and TLR3 signaling. 16,17 In addition, HCV proteins have been shown to upregulate TLR2 and TLR4 expression on Raji cells and peripheral blood mononuclear cells, thereby modulating the cell's pro-inflammatory response. 18,19 Moreover, several studies by our group and others have provided evidence that the HCV core can activate TLR2 on human monocytes, macrophages and Kupffer cells, which induces production of inflammatory cytokines by activating the MyD88-dependent TLR signaling pathway.…”

Sensing and Control of Bluetongue Virus Infection in Epithelial Cells via RIG-I and MDA5 Helicases

“…RIG-I and MDA5 are activated upon the detection of viral RNA molecules. The mechanisms of this activation have been largely characterized and depend on specific RNA features (19,25,31,45,53,54). RIG-I preferentially recognizes RNA sequences containing 5= triphosphorylated (5=ppp) ends, which represents a nonself RNA PAMP (19).…”