The sensor RIG-I detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to have a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during infection with a DNA virus are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bound to RIG-I during infection with herpes simplex virus 1 (HSV-1). Infection with HSV-1 induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated the abundance of RNA5SP141-interacting proteins, which allowed RNA5SP141 to bind RIG-I and induce the expression of type I interferons. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related virus Epstein-Barr virus (EBV), as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following depletion of their respective binding proteins by the virus.
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We thank B. Roizman (University of Chicago) for HSV-1Δvhs mutant and revertant; Y. Kawaguchi (University of Tokyo) for the bacterial artificial chromosome clone of HSV-1; A. Garcia-Sastre (Mount Sinai) for IAV wild-type and ΔNS1 recombinant virus; N. Raab-Traub (UNC-Chapel Hill) for AGS-EBV cells; D. Knipe (Harvard University) for the antibody to ICP8; W. Azab for help with manuscript preparation; and K. Waraska and A. Diallo for discussions. Supported by the US National Institutes of Health (R21 AI133361 and R01 AI087846 to M.U.G.), the German Research Foundation (fellowship SP 1600/1-1 to K.M.J.S.; and HO2489-8 to K.-P.H.) and BioSysNet (K.-P.H. and C.L.).
The authors declare no competing financial interests.
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(a) Left, middle left: qRT-PCR analysis of IFNB1 and TNF mRNA in primary NHLF cells transfected with either non-targeting control siRNA (si.Ctrl) or RIG-I-, cGAS-, or IFI16-specific siRNAs (si.RIG-I, si.cGAS, si.IFI16) for 30 h and then infected with HSV-1 (MOI 0.1) for the indicated times. Results were normalized to 18S rRNA, and fold induction is shown relative to mock-infected control cells. Right three panels: Knockdown efficiency of endogenous RIG-I (DDX58), cGAS (MB21D1) and IFI16 was confirmed by qRT-PCR. (b) qRT-PCR analysis of IFNB1, IFIH1, OASL1, RSAD2, IFIT2, and CCL5 mRNA in Ddx58 –/– and Ddx58 +/+ MEFs infected with HSV-1 (MOI 1 each) for the indicated times. Results were normalized and shown as in (a). Data represent mean and s.d. of n = 3 biological replicates, and are representative of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t-test); NS, statistically not significant; ND, not detected.
(a) Equal pulldown (PD) of FLAG–RIG-I and FLAG-GFP from the experiment shown in Figures 1a and 1b was confirmed by immunoblot (IB) with anti-FLAG. (b) qRT-PCR analysis of IFNB1 mRNA in primary NHLF cells transfected for 20 h with RNA purified by streptavidin-PD from total cellular RNA using 3′-biotinylated locked nucleic acid (LNA) oligos specific for RNA5SP141. RNAs purified using LNA oligos designed to capture a nonsense ‘scramble’ sequence and the highly abundant small nuclear RNA RNU1-1 that is not known to be immunostimulatory served as negative controls. (c) Validation of the LNA oligos targeting RNA5SP141 (LNA_RNA5SP141), RNU1-1 (LNA_RNU1-1), or ‘scramble’ RNA (LNA_Scramble). Representative enrichment of RNU1-1 and RNA5SP141 by streptavidin-PD as described in (b). Data are representative of two independent experiments (mean and s.d. of n = 3 biological replicates in (b), n = 3 technical replicates in (c)). **P < 0.01 (unpaired t-test). NS, statistically not significant.
(a) Predicted secondary structure of RNA5SP141 modeled using the Vienna RNAfold web server. MFE, minimum free energy. 5′-ppp, 5′-triphosphate. (b) Forward (F) and reverse (R) DNA oligonucleotides used to construct in vitro transcription templates for RNA5SP141, parental 5S rRNA, and ‘scrambled’ RNA. (c) Virtual gel image of the indicated in vitro–transcribed RNAs analyzed by Agilent 2100 Bioanalyzer. (d) IFNB1 transcripts in primary NHLF cells that were either mock-transfected or transfected with the indicated amounts of in vitro–transcribed RNA5SP141 or RABVLe (positive control), determined by qRT-PCR analysis at 18 h post-transfection. Data in (d) represent mean and s.d. of n = 3 biological replicates, and are representative of three independent experiments.
(a) Left: qRT-PCR analysis of IFNB1 mRNA in primary NHLF cells transfected with either non-targeting control siRNA (si.Ctrl), or siRNAs targeting RIG-I (si.RIG-I) or MDA5 (si.MDA5), followed 30 h later by transfection for 16 h with 1 pmol of the indicated in vitro–transcribed RNAs, or 0.05 μg/ml HMW-poly(I:C) which served as a control. Middle, right: Knockdown efficiency of endogenous RIG-I (DDX58) and MDA5 (IFIH1) was confirmed by qRT-PCR. (b) Relative expression of RNA5SP141 in HEK 293T cells infected with HSV-1WT (MOI 1) for 16 h, as compared to uninfected cells, determined by RNAseq analysis. Red boundaries represent ± 2-fold change in gene expression. (c) qRT-PCR analysis of RNA5SP141 transcripts in primary NHLF cells infected with HSV-1WT (MOI 1) for the indicated times. (d,e) qRT-PCR analysis of IL6 (d) and IL8 (e) transcripts in primary NHLF cells that were mock-infected or infected with HSV-1Δvhs or HSV-1WT (revertant) (MOI 10 each) for 16 h. Results were normalized to 18S rRNA, and fold induction is shown relative to values for mock-infected control cells. Data are representative of at least two independent experiments (mean and s.d. of n = 3 biological replicates in (a) and (c-e)).
Supplementary Figure 5 Depletion of RNA5SP141 dampens the cytokine response induced by infection with HSV-1 but not that induced by infection with SeV.
(a) Left: qRT-PCR analysis of TNF mRNA in primary NHLF cells transfected with either non-targeting control siRNA (si.Ctrl), or siRNAs targeting RIG-I (si.RIG-I) or RNA5SP141 (si.5SP141) for 72 h and then infected with HSV-1WT (MOI 0.1) for 16 h. Middle, right: Knockdown efficiency of endogenous RIG-I (DDX58) and RNA5SP141 was confirmed by qRT-PCR. (b) ELISA of IFN-β (left) and CCL5 (right) in the supernatants of primary NHLF cells that were transfected with either non-targeting control siRNA (si.Ctrl), or siRNAs targeting RIG-I (si.RIG-I) or RNA5SP141 (si.5SP141) for 72 h, followed by infection with SeV (50 HAU/ml) for 24 h. Data are representative of two independent experiments (mean and s.d. of n = 3 biological replicates in (a), and n = 2 biological replicates in (b)). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t-test). NS, statistically not significant; ND, not detected.
Representative knockdown efficiency of endogenous RIG-I (DDX58) (left) and RNA5SP141 (right) in primary NHLF cells achieved by transfection of two different gapmers targeting RIG-I (Gap RIG-I_1 and Gap RIG-I_2) or RNA5SP141 (Gap 5SP141_1 and Gap 5SP141_2), determined by qRT-PCR at 72 h post-transfection. Non-targeting control gapmer (Gap NT) served as control. Data represent mean and s.d. of n = 3 technical replicates, and are representative of two independent experiments.
(a-d) IFNB1, TNF, IL6, and IL8 transcripts in AGS-EBV cells treated with 2.5 mM sodium butyrate (NaB) for the indicated times to induce EBV reactivation, assessed by qRT-PCR analysis. (e) Efficient EBV reactivation was confirmed by determining the transcript amounts of EBV early gene BMRF1 (EA-D) by qRT-PCR analysis. Data represent mean and s.d. of n = 3 technical replicates.
Supplementary Figure 8 Silencing of RNA5SP141 diminishes the antiviral response induced by infection with IAV.
(a) Representative knockdown efficiency of RIG-I (DDX58) and RNA5SP141 in primary NHLF cells achieved by transfection of siRNAs targeting RIG-I (si.RIG-I) or RNA5SP141 (si.5SP141), assessed by qRT-PCR at 72 h post-transfection. (b) IFNB1, IFIT2, and CCL5 transcripts in HEK 293T cells transfected with non-targeting control siRNA (si.Ctrl), or siRNAs targeting RIG-I or RNA5SP141 (si.RIG-I or si.5SP141) followed by infection with IAV (MOI 0.1) for 16 h, determined by qRT-PCR. (c) Representative knockdown efficiency of RIG-I (DDX58) and RNA5SP141 in HEK 293T cells achieved by transfection of siRNAs targeting RIG-I (si.RIG-I) or RNA5SP141 (si.5SP141), assessed by qRT-PCR at 72 h post-transfection. (d) qRT-PCR analysis of IFNB1 mRNA in HEK 293T cells transfected with siRNAs as in (b) and then infected with IAVΔNS1 (MOI 0.1) for 16 h. Data represent mean and s.d. of n = 3 technical replicates (a,c) or n = 3 biological replicates (b,d), and are representative of two (a-c) or three (d) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t-test).
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Chiang, J.J., Sparrer, K.M.J., van Gent, M. et al. Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I-mediated immunity. Nat Immunol 19, 53–62 (2018). https://doi.org/10.1038/s41590-017-0005-y
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