Interferon modulation of cellular microRNAs as an antiviral mechanism

Abstract

RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive. Here we show that interferon beta (IFNbeta) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNbeta-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNbeta-induced miRNAs reproduces the antiviral effects of IFNbeta on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNbeta against HCV. In addition, we demonstrate that IFNbeta treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication. Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections.

Figure 1. Regulation of miRNA expression by IFNβ in Huh7 cells and primary hepatocytes

a, b, Huh7 cells (a) or primary hepatocytes (b) were stimulated with 100 U ml⁻¹ IFNβ for 2 h, and the indicated miRNAs were quantified by qPCR. ISG54 induction is shown for comparison. c, Time course of miRNA induction by IFNβ: Huh7 cells were stimulated with 100 U ml⁻¹ IFNβ for the indicated times, and miR-1, miR-196 or ISG54 expression was quantified by qPCR. d, Dose—response analysis of miRNA induction by IFNβ: Huh7 cells were stimulated with the indicated doses of IFNβ for 2 h, and miR-1, miR-196 or ISG54 expression was quantified by qPCR. e, Time course and dose—response analysis of miR-122 downregulation by IFNβ: Huh7 cells were stimulated as described in c and d, and miR-122 was quantified by qPCR. Error bars, means ± s.d. of at least four independent experiments.

Figure 2. IFNβ-induced miRNAs display anti-viral activity against HCV

a, JFH1-replicon-containing Huh7 cells were transfected with single non-specific miRNA-mimics (miR-ctrl), a pool of control miRNAs (miR-ctrl(mix5)) or anti-miRNAs (anti-miR-ctrl), or specific miRNA-mimics corresponding to the eight IFNβ-induced miRNAs, or specific anti-miRNAs. In addition, a combination of the five miRNAs that displayed anti-viral activity individually was used (miR-mix5) with or without anti-miR-122, as indicated, and HCV RNA was quantified by qPCR after 48 h. b, Same as a, except Huh7 cells were infected with live JFH-1 virus for 48 h (error bars, means ± s.d. of at least four independent experiments; P values are from paired Student’s t-tests).

Figure 3. IFNβ-induced miRNAs directly target viral genomic RNA

a, b, An infectious chimaeric virus was constructed from JFH1 and J6CF as shown in a; the numbers in brackets indicate the location in the virion (nucleotides) that the miRNA binds. The Huh7-cell subclone Huh7.5.1c2 was transfected with either miR-196 or miR-448, or with the mutant miR-196* and miR-448* harbouring a compensatory mutation in the seed sequence as outlined in a. Transfected Huh7.5.1c2 cells were infected with JFH1(D183) (ref. 23) or chimaeric J6/JFH, and HCV RNA was quantified by qPCR after 24 or 60 h post infection, respectively, during the phase of exponential viral RNA amplification, to accommodate the difference in replication kinetics between the two viruses (error bars, means ± s.e.m. of 8 independent experiments; P values are from paired Student’s t-tests).

Figure 4. IFNβ-induced miRNAs mediate anti-viral IFNβ responses against HCV

JFH1-replicon-containing Huh7 cells were transfected with non-specific miRNA-mimics (miR-ctrl), or non-specific anti-miRNAs (anti-miR-ctrl), or a pool of anti-miRNAs (anti-miR-ctrl(5)), or a combination of anti-miRNA complementary to the five IFNβ-induced miRNAs and with potent anti-viral effect (anti-miRNA-mix5), and/or with specific miRNAs and anti-miRNAs, as indicated, before stimulation with IFNβ for 48 h. HCV RNA was quantified by qPCR (means ± s.d. of at least four independent experiments; P values are from paired Student’s t-tests).