Nonsegmented Negative-Sense RNA Viruses Utilize N6-Methyladenosine (m6A) as a Common Strategy To Evade Host Innate Immunity

Abstract

N⁶-Methyladenosine (m⁶A) is the most abundant internal RNA modification catalyzed by host RNA methyltransferases. As obligate intracellular parasites, many viruses acquire m⁶A methylation in their RNAs. However, the biological functions of viral m⁶A methylation are poorly understood. Here, we found that viral m⁶A methylation serves as a molecular marker for host innate immunity to discriminate self from nonself RNA and that this novel biological function of viral m⁶A methylation is universally conserved in several families in nonsegmented negative-sense (NNS) RNA viruses. Using m⁶A methyltransferase (METTL3) knockout cells, we produced m⁶A-deficient virion RNAs from the representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae and found that these m⁶A-deficient viral RNAs triggered significantly higher levels of type I interferon compared to the m⁶A-sufficient viral RNAs, in a RIG-I-dependent manner. Reconstitution of the RIG-I pathway revealed that m⁶A-deficient virion RNA induced higher expression of RIG-I, bound to RIG-I more efficiently, enhanced RIG-I ubiquitination, and facilitated RIG-I conformational rearrangement and oligomerization. Furthermore, the m⁶A binding protein YTHDF2 is essential for suppression of the type I interferon signaling pathway, including by virion RNA. Collectively, our results suggest that several families in NNS RNA viruses acquire m⁶A in viral RNA as a common strategy to evade host innate immunity.IMPORTANCE The nonsegmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There are no vaccines or antiviral drugs for many of these viruses. We found that representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae among the NNS RNA viruses acquire m⁶A methylation in their genome and antigenome as a means to escape recognition by host innate immunity via a RIG-I-dependent signaling pathway. Viral RNA lacking m⁶A methylation induces a significantly higher type I interferon response than m⁶A-sufficient viral RNA. In addition to uncovering m⁶A methylation as a common mechanism for many NNS RNA viruses to evade host innate immunity, this study discovered a novel strategy to enhance type I interferon responses, which may have important applications in vaccine development, as robust innate immunity will likely promote the subsequent adaptive immunity.

Keywords: N6-methyladenosine; innate immunity; negative-strand RNA virus.

FIG 1

The VSV RNAs are m⁶A methylated. (a and b), Distribution of m⁶A peaks in the VSV antigenome (a) and genome (b). A schematic diagram of the VSV antigenome containing all genes (N, P, M, G, and L) is shown. Total RNAs were extracted from purified VSV virions grown in A549 cells and were analyzed by m⁶A immunoprecipitation (IP) followed by m⁶A-seq. Shaded areas show the distribution of m⁶A immunoprecipitation reads mapped to the VSV antigenome (a) or genome (b). The baseline signal from input samples is shown as a line. (c) Distribution of m⁶A peaks in the VSV mRNAs. Polyadenylated mRNAs were isolated from VSV-infected A549 cells and subjected to m⁶A-seq. Shaded pink areas show the distribution of m⁶A immunoprecipitation reads mapped to the VSV mRNAs. The baseline signal from input samples is shown as a line. Data are presented as the averages from two independent virus-infected A549 cell samples (n = 2).

FIG 2

The SeV RNAs are m⁶A methylated. (a and b), Distribution of m⁶A peaks in the SeV antigenome/mRNAs (a) and genome (b). A schematic diagram of the rSeV-GFP antigenome containing all genes (N, P, M, GFP, F, HN, and L) is shown. Total RNA was extracted from rSeV-GFP or mock-infected A549 cells and subjected to sonication. RNA containing m⁶A methylation was pulled down by m⁶A antibody, followed by m⁶A-seq. The shaded pink areas show the distribution of m⁶A immunoprecipitation (IP) reads mapped to the SeV antigenome/mRNAs (a) or genome (b). The baseline signal from input samples is shown as a line. Data presented in panels a and b are the averages from three independent virus-infected A549 cell samples (n = 3). (c) Distribution of m⁶A peaks in the SeV genome in individual replicates.

FIG 3

The viral RNA of virus grown in METTL3 knockout U2OS cells is defective in m⁶A methylation. (a) Western blot showing METTL3 expression in METTL3-knockout U2OS cells and wild-type U2OS cells. (b to d) Quantification of m⁶A level in virion RNA. Shown are the relative m⁶A levels in virion RNAs from SeV (b), MeV (c), hMPV (d), and VSV (e) grown on METTL3 KO/WT U2OS cells. Each virus was purified through 30 to 50% linear sucrose gradient ultracentrifugation. Virion RNA was extracted, and the total m⁶A level of each virion RNA was quantified by m⁶A RNA methylation assay. (f) Total viral RNA from VSV-infected METTL3 KO U2OS cells is defective in binding to m⁶A antibody by MeRIP assay. An MeRIP assay was carried out to determine the binding of RNA to m⁶A antibody using the Magna MeRIPTM m⁶A kit. Anti-m⁶A antibody was first conjugated to magnetic beads. Total RNA (15 μg) was extracted from VSV-infected METTL3 KO/WT U2OS cells and incubated with m⁶A antibody-associated beads at 4°C for 2 h with rotation. The RNA-associated magnetic beads were then washed for 3 times. Total RNA was extracted from beads by TRIzol reagent and quantified by real-time RT-PCR using primers annealing to VSV antigenome and genome. Data shown are the mean ± standard deviation (SD) from n = 3 (b, e, and f), n = 6 (c), or n = 4 (d) biologically independent experiments. Statistical significance was determined by two-sided Student's t test: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

m⁶A-deficient viruses and their viral RNAs induce higher type I IFN production. (a to c) Comparison of IFN production triggered by virion RNAs of SeV (a), MeV (b), and hMPV (c) produced from METTL3 KO or WT U2OS cells. A549 cells were transfected with 10⁵ RNA copies of SeV (a), 5 × 10⁶ RNA copies of MeV (b), and 10⁷ RNA copies of hMPV (c), with each virus grown on METTL3 KO or WT U2OS cells. IFN-β production was measured by an ELISA kit at indicated time points. (d to e) IFN-β response in A549 cells transfected with total RNA from VSV-infected cells. Total RNA was extracted from VSV-infected METTL3 KO or WT U2OS cells, and the antigenome/genome was quantified by real-time RT-PCR. A549 cells were transfected with 10⁸ (d) and 10⁷ (e) copies of viral RNA. IFN-β was measured by ELISA at indicated time points. (f to h) IFN-β mRNA level in A549 after viral infection with or without cycloheximide (CHX) treatment. A549 cells were treated for 1 h with 0 or 50 μg/ml CHX and then infected with either SeV (f), MeV (g), or VSV (h) grown in METTL3 KO U2OS or WT U2OS cells. Total RNA was extracted from virus-infected cells, and IFN-β mRNA was quantified by real-time RT-PCR. The relative mRNA level between METTL3 KO- and WT U2OS cell-derived viruses was calculated. The data shown are the mean ± SD from n = 3 biologically independent experiments. Statistical significance was determined by two-sided Student's t test: *, P < 0.5; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

IFN response in A549 knockout cells transfected with m⁶A-deficient viral RNA. (a to l) IFN production after transfection of virion RNA. Confluent wild-type (a, e, i), MDA5 knockout (b, f, j), RIG-I knockout (c, g, k), or MAVS knockout (d, h, l) A549-Dual cells were transfected with the same amounts of SeV (a to d, 10⁵ RNA copies), MeV (e to h, 2 × 10⁵ RNA copies), and hMPV (i to l, 10⁷ RNA copies) virion RNAs from either METTL3 KO or WT cells. (m to p) IFN production after transfection of total RNA from VSV-infected cells. Confluent wild-type (m), MDA5 knockout (n), RIG-I knockout (o), or MAVS knockout (p) A549-Dual cells were transfected with the same amounts of total RNA from VSV-infected METTL3 KO cells or METTL3 WT cells containing 5 × 10⁶ copies of viral RNA. Cell culture supernatants were harvested at 16 and 24 h after inoculation, IFN-β in the supernatant at indicated time points was measured by a commercial ELISA kit. Data shown are the mean ± SD from n = 3 biologically independent experiments. Statistical significance was determined by two-sided Student's t test: *, P < 0.5; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

m⁶A-deficient viral RNA enhances expression of molecules involved in type I IFN signaling pathway. (a to c), m⁶A-deficient virion RNA increases expression of RIG-I and MDA5 and induces higher IRF3 phosphorylation. A549 cells were transfected with virion RNA of SeV (a, at doses of 2 × 10⁵, 1 × 10⁵, and 5 × 10⁴ RNA copies), MeV (b, at doses of 1 × 10⁶, 5 × 10⁵, and 2 × 10⁵ RNA copies), and hMPV (c, at doses of 10⁷, 10⁶, and 10⁵ RNA copies) grown on METTL3 KO or WT U2OS cells. (d) m⁶A-deficient total RNA from VSV-infected cells increases expression of RIG-I and MDA5 and induces higher IRF3 phosphorylation. A549 cells were transfected with total RNA from VSV-infected cells at doses of 1 × 10⁸ and 1 × 10⁷ RNA copies grown on METTL3 KO or WT U2OS cells. At indicated times, cell lysates were analyzed by Western blotting using antibodies specific to RIG-I, MDA5, IRF3, IRF3 phosphorylated at S386, or β-actin. Western blots are representative of n = 3 biologically independent experiments. (e) A cartoon model for type I IFN signaling pathway. m⁶A-sufficient or -deficient viral RNA is detected by either RIG-I or MDA5, engaging the adaptor protein MAVS, which leads to the phosphorylation of IRF‐3 by TBK1/inducible IκB kinase (IKK‐i), the formation of IRF‐3 homodimers and/or heterodimers, and translocation into the nucleus, resulting in the expression of type I IFNs.

FIG 7

m⁶A-deficient virion RNA increases RIG-I binding. Shown are the results from an affinity binding assay of RIG-I with each virion RNA. RIG-I-conjugated magnetic beads were incubated with virion RNA purified from METTL3 KO or WT U2OS cells. (a) One aliquot of beads was analyzed by Western blotting. (b to e) Antigenome or genome RNA bound to magnetic beads was quantified by real-time RT-PCR. Results were normalized as the ratio between immunoprecipitated RNA from METTL3 KO cells and that from METTL3 WT cells. The data shown are the mean ± SD from n = 3 (b to d) or n = 6 (e and f) biologically independent experiments. Statistical significance was determined by two-sided Student's t test: *, P < 0.5, **, P < 0.01.

FIG 8

m⁶A-deficient virion RNA facilitates RIG-I:RNA conformation change and increases the ubiquitination of RIG-I. (a) Analysis of RIG-I:RNA complex in native PAGE gel. Purified RIG-I was incubated with poly(I·C) or 10⁷ copies of virion RNA in MOPS buffer in the presence of RNase inhibitor and AMP-PNP (2 mM). The reaction mixtures were incubated at 37°C for 30 min to enable RIG-I:RNA complex formation. Ten microliters of the mixture was mixed with an equal volume of native PAGE buffer, and RIG-I:RNA complex was separated in native PAGE gel, followed by Western blotting with anti-RIG-I helicase antibody to detect the RIG-I:RNA complex. Gels with short and long exposures are shown. The Western blots shown are the representatives of three independent experiments. (b) Analysis of RIG-I:RNA conformation by limited trypsin digestion in denaturing SDS-PAGE. The RIG-I:RNA complex was formed as described for panel a. Limited trypsin digestion of RIG-I protein in the absence of RNA ligand for 0 to 2 h (left panel) or in the presence of poly(I·C) or viral RNA (right panel) for 2 h is shown. The Western blots shown are the representatives of three independent experiments. (c) In vitro ubiquitination analysis of RIG-I. A 1.0 μM concentration of purified RIG-I was incubated with 1 ng/μg of 42-bp dsRNA and different doses of SeV virion RNA from METTL3 KO or WT U2OS cells. Ubiquitination of RIG-I was analyzed by anti-RIG-I blotting. The blots shown are the representatives of three independent experiments.

FIG 9

YTHDF2 is essential for suppression of the IFN signaling pathway. (a) Western blot analysis of YTHDF2 in YTHDF2 knockout A549 cells and control sgRNA-treated A549 cells. (b) SeV infection induces a higher IFN signaling pathway in YTHDF2 knockout A549 (A549-Y2 KO) cells compared to WT A549 cells. Confluent cells were infected by SeV at an MOI of 1.0, and cell lysates were analyzed by Western blotting at the indicated time points. (c) IFN production in WT and YTHDF2 knockout A549 cells upon SeV infection at an MOI of 1.0. (d) hMPV infection induces a higher IFN signaling pathway in A549-Y2 KO cells compared to A549 WT cells. Cells were infected by hMPV at an MOI of 1.0. (e) IFN production in WT and YTHDF2 knockout A549 cells upon hMPV infection at an MOI of 1.0. (f) Comparison of IFN signaling pathways in A549-Y2 KO cells and A549 WT cells upon transfection of equal amounts (10⁶ RNA copies) of WT SeV RNA and m⁶A-deficient SeV RNA. (g) IFN production in WT and YTHDF2 knockout A549 cells upon SeV RNA transfection (10⁶ RNA copies). (h) Comparison of IFN signaling pathway in A549-Y2 KO cells and A549 WT cells upon transfection of equal amounts (10⁶ RNA copies) of WT hMPV RNA and m⁶A-deficient hMPV RNA. (i) IFN production in WT and YTHDF2 knockout A549 cells upon hMPV RNA transfection (10⁶ RNA copies). (j) Comparison of IFN signaling pathways in A549-Y2 KO cells and A549 WT cells upon transfection of equal amounts (10⁶ RNA copies) of WT hMPV RNA and hMPV G1-14 RNA. (k) IFN production in WT and YTHDF2 knockout A549 cells upon WT hMPV RNA and hMPV G1-14 RNA transfection (10⁶ RNA copies). The Western blots shown are the representatives of three independent experiments. The interferon data shown are the mean ± SD from n = 3 biologically independent experiments. Statistical significance was determined by two-sided Student's t test: *, P < 0.5; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 10

Model for how m⁶A methylation invades host innate immunity in NNS RNA viruses. The newly synthesized genome and antigenome of NNS RNA viruses are first methylated by m⁶A writer proteins, which are encapsidated by N protein to serve as a template for replication and transcription. Some of the genome and antigenome may not be encapsidated at an early stage in the virus replication cycle, particularly when the concentration of N protein is low. These unmodified antigenomes or genomes would be recognized by RIG-I to induce the IFN signaling pathway. m⁶A methylation in genome and antigenome prevents RIG-I binding, conformational change, oligomerization, and K63-linked polyubiquitination, which result in lower activation of MAVS and subsequent phosphorylation of IRF3, thereby inhibiting type I IFN production. In addition, YTHDF2 binds to the m⁶A-methylated genome and antigenome, which suppresses type I IFN signaling.