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N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I

Abstract

Internal N6-methyladenosine (m6A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m6A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m6A methylated and that viral m6A methylation promotes HMPV replication and gene expression. Inactivating m6A addition sites with synonymous mutations or demethylase resulted in m6A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m6A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m6A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m6A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m6A can serve as a target to attenuate HMPV for vaccine purposes.

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Fig. 1: The HMPV RNAs are m6A methylated and m6A methylation promotes HMPV replication.
Fig. 2: m6A-deficient rHMPVs and their virion RNAs induce higher type I IFN responses.
Fig. 3: IFN response and NF-κB activation in A549 cells infected with m6A deficient HMPVs or transfected with m6A deficient virion RNA.
Fig. 4: m6A-deficient HMPVs and virion RNA induce a higher expression of RIG-I.
Fig. 5: m6A-deficient virion RNA increases RIG-I binding affinity and facilitates RIG-I:RNA conformation change.
Fig. 6: Replication, interferon response, pathogenicity and immunogenicity of m6A-deficient rHMPVs.

Data availability

The data supporting the findings of this study are available with the article and its Extended Data and Supplementary Information files, or are available from the corresponding author upon request. The raw sequencing data obtained from the MeRIP-seq reported in this paper has been deposited at the Gene Expression Omnibus with accession no. GSE136139. The source data underlying Figures, Extended Data Figures and Supplementary Figures are provided as a Source Data file.

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Acknowledgements

This study was supported by grants from the National Institutes of Health (no. R01AI090060) to J.L., no. P01 AI112524 to M.E.P., S.N. and J.L., and nos. R01 HG008688 and RM1 HG008935 to C.H. C.H. is an investigator of the Howard Hughes Medical Institute. We thank R. A. M. Fouchier for the infectious cDNA clone of HMPV, J. Yount for the RIG-I plasmid and members of the J. Li laboratory for critical readings of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

M.L. carried out most of the virological, biochemical and animal experiments. Z.Z. performed m6A–seq and analysed all m6A data, with help from B.S.Z. M.X. measured m6A levels in host and viral RNA. A.L. performed the [35S]-methionine metabolic labelling experiment. S.N., O.H. and X. L. designed and helped with animal experiments. T.Z.G. performed some of mutagenesis experiments. M.E.P., Y.X. and Z.F. generated biochemical reagents. J.Z. helped with histology. C.H. supervised m6A experiments, analysed the data and interpreted the results. M.E.P. and S.N. contributed to supervision and data analysis. J.L. directed the project, analysed the data and wrote the paper with help from all of the authors.

Corresponding author

Correspondence to Jianrong Li.

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Competing interests

C.H. is a scientific founder of Accent Therapeutics. J.L., C.H., M.E.P. and S.N. have filed a provisional patent application (no. 62/748,175).

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Extended data

Extended Data Fig. 1 HMPV genome and antigenome are packaged into virion.

Highly purified HMPV virions were disrupted by detergent, digested with RNase, and the RNase-resistant viral nucleocapsid (N-RNA complex) was pulled down by HMPV N antibody. Both genome and antigenome were detected in N-RNA complex by real-time RT-PCR (a-c). This demonstrates that both the genome and the antigenome are indeed encapsidated by N protein and packaged into HMPV virions. (a), Quantification of genome and antigenome in HMPV virions by real-time RT-PCR. Wild type HMPV was grown in T150 flasks of A549 cells and cell culture supernatant was collected at 40 hpi. HMPV was purified by 20% sucrose cushion, the virion pellet was resuspended in 100 µL NTE buffer, treated with RNase at room temperature for 30 min.The reaction system was diluted in 5 ml NTE buffer and the virion was pelleted down in SW55 Ti ultracentrifugation tube on 10% sucrose cushion and resuspended in 100 µL NTE buffer. (b), Pull down of N-RNA complex by N antibody. RNase-treated virion was disrupted by 10 × Disruption Buffer, mixed with Protein A/G Magnetic beads (MilliPore, LSKMAGAG02) bound with mouse anti-HMPV N antibody (MiiliPore, MAB80138) and incubated at room temperature for 2 h and washed with TBS buffer for 3 times. The beads were then subjected for Western blot. (c), Quantification of genome and antigenome in N-RNA complex by real-time RT-PCR. Total RNA was extracted from N-complex pulled down by magnetic beads and subjected to real-time RT-PCR. All results are from three independent experiments. SDS-PAGE and Western blots (b), shown are the representatives of n = 3 biologically independent experiments. Viral RNA copies (a and c) are the geometric mean titers (GMT) of n = 3 biologically independent experiments ± standard deviation.

Source data

Extended Data Fig. 2 HMPV infection alters the transcriptome of host transcripts.

Total RNAs were isolated from mock-infected and HMPV-infected A549 cells. Poly(A) enriched mRNAs were purified and subjected to m6A-seq. (a), Motif analysis to identify consensus sequences for m6A methylation sites in uninfected and HMPV-infected A549 cells. Frequency of nucleotides at the three positions flanking the central m6A sites is shown. High quality m6A peaks were detected in both HMPV-infected and mock-infected samples, as demonstrated by finding the m6A consensus sequence GGACU similarly enriched in both sets of samples. (b), Metagene analysis of normalized m6A peak distribution along the human reference mRNA in control and infected cells. Metagene analysis showed that HMPV infection slightly increased m6A levels in the 5’ and 3’ UTR regions of the host transcriptome, but slightly decreased the m6A levels in the coding sequence (CDS) and noncoding sequence (NCS) regions of the host mRNAs. (c and d) GO graphs showing functional clusters from upregulated genes (c) or downregulated genes (d) identified in HMPV-infected cells. GO analysis revealed that the upregulated genes are strikingly enriched in innate host defense transcripts including the cytokine and interferon signaling pathway and inflammatory responses (c). Numerous interferon encoded genes are upregulated including interferon lambda receptor 1, interferon beta 1, interferon lambda 2, interferon lambda 4, and genes involved in pattern recognition receptor (PRR) including RIG-I, MDA5, LPG2, and multiple interferon-stimulated genes (ISGs). In contrast, the downregulated genes are enriched in the cell cycle, metabolism, and translation category (d).

Extended Data Fig. 3 Overexpression of m6A reader and writer proteins increases HMPV RNA syntheses in A549 cells.

(a), Overexpression of m6A reader proteins. A549 cells were transfected with plasmids encoding YTHDF1, 2, 3, or YTHDC1. At 24 h post-transfection, cells were lysed and subjected to Western blot. YTHDF1-3 proteins were detected by anti-HA-tag antibody and YTHDC1 was detected by anti-YTHDC1 antibody. (b), The effects of overexpression of m6A reader proteins on HMPV genome replication. A549 cells were transfected with plasmids encoding YTHDF1, 2, 3, or YTHDC1. At 24 h post-transfection, cells were infected with rHMPV at an MOI of 5.0. At 12, 18, 24, and 48 h post-infection, total RNA was purified from rHMPV-infected cells using TRizol, and genomic RNA was quantified by real-time RT-PCR using specific primers annealing to the HMPV leader sequence and N gene. (c), The effects of overexpression of m6A reader proteins on HMPV antigenome replication. HMPV antigenome was quantified with specific primers annealing to the HMPV trailer sequence and L gene. (d), The effects of overexpression of m6A reader proteins on G mRNA transcription. N- and G-mRNA and GAPDH mRNA copies were quantified from cDNA pool generated from total RNA and Oligo (dT)23. RNA and mRNA copies were normalized by GAPDH. (e), The effects of overexpression of m6A reader proteins on N mRNA transcription. (f-i), The effects of overexpression of m6A writer proteins on HMPV genome RNA replication (f), antigenome RNA replication (g), G mRNA synthesis (h), and N mRNA synthesis (i). A549 cells were transfected with plasmids encoding HA-tagged METTL3 and/or METTL14. At 24 h post-transfection, cells were infected with rHMPV at an MOI of 5.0. At 12, 18, 24, and 48 h post-infection, total RNA was purified from rHMPV-infected cells, and genome RNA, antigenome RNA, N mRNA, and G mRNA were quantified by real-time RT-PCR. All results are from n = 3 biologically independent experiments. Western blots (a) shown are the representatives of n= 3 biologically independent experiments. RNA copies (b-i) are the geometric mean titers (GMT) of n = 3 biologically independent experiments ± standard deviation. Statistical significance was determined by two-sided student’s t-test. Exact P values are included in Data Source. *P < 0.05, **P < 0.01, and ***P < 0.001.

Source data

Extended Data Fig. 4 YTHDF1, 2, 3 (reader) proteins promote HMPV replication, gene expression, and progeny virus production in HeLa cells.

(a), Detection of YTHDF1, 2, 3 in HeLa cells stably overexpressing YTHDF1, 2, or 3. Western blot confirmed the overexpression of YTHDF1-3 proteins in HeLa cells using anti-Flag antibody. (b), YTHDF1, 2, 3 enhance HMPV protein expression in HeLa cells. HeLa cells stably overexpressing these YTHDF proteins were infected with rgHMPV (rHMPV expressing GFP) at an MOI of 0.5. Total cell extracts were harvested from HMPV-infected HeLa cells at the indicated times and subjected to Western blot using antibody against HMPV N, F, or G protein. (c), YTHDF1, 2, 3 enhance GFP expression in HMPV-GFP-infected cells. HeLa cells stably overexpressing these YTHDF proteins were infected with rgHMPV at an MOI of 1.0, and GFP expression was monitored at the indicated times by fluorescence microscopy. (d), YTHDF1, 2, 3 increase the number of GFP-positive cells quantified by flow cytometry. Cells from panel c were trypsinized, fixed in 4 % of paraformaldehyde, and the number of GFP-positive cells quantified by flow cytometry. The mock-infected cells (GFP negative) were used for gating controls. The number of GFP-positive and negative cells was sorted. (e), YTHDF1, 2, 3 enhance GFP intensity. The intensity of GFP signal was determined by flow cytometry. (f), YTHDF1, 2, 3 increase HMPV progeny virus production. The release of infectious HMPV particles was monitored by a single-step growth curve. Virus titer was measured by an immunostaining plaque assay. (g), HMPV genome RNA replication. (h), HMPV antigenome RNA replication. (i), G mRNA synthesis. (i) N mRNA synthesis. All results are from n = 3 biologically independent experiments. Western blots (a and b) and GFP images (c) shown are the representatives of n = 3 independent experiments. Flow cytometry data (d and e) are the means of n = 3 independent experiments ± standard deviation. All RNA data were quantified by real-time RT-PCR. RNA copies (g-j) and viral titers (f) are the geometric mean titers (GMT) of n = 3 independent experiments ± standard deviation. Statistical significance was determined by two-sided student’s t-test. Exact P values are included in Data Source. *P < 0.05, **P < 0.01, and ***P < 0.001.

Source data

Extended Data Fig. 5 Knockdown of m6A eraser proteins enhances HMPV gene expression.

(a), Western blot of HMPV proteins. A549 cells were transfected with siRNA targeting FTO and/or ALKBH5. At 24 h post-transfection, cells were infected with rHMPV at an MOI of 0.5. At 12, 18, and 24 h post-infection, cell lysates were harvested for Western blot analysis using HMPV antibody. Knockdown was confirmed by Western blot using anti-FTO and anti-ALKBH5 antibody. (b), Quantification of HMPV G protein. The density of G protein in Western blot was quantified by ImageJ. Data are averages of three independent experiments. (c), Quantification of HMPV N protein. The density of N protein in Western blot was quantified by Image J. Data are average of three independent experiments. (d), Knockdown m6A eraser proteins increases HMPV progeny virus production. The release of infectious HMPV particles was monitored by a single-step growth curve. Virus titer was measured by an immunostaining plaque assay. (e), HMPV genome RNA replication. (f), HMPV antigenome RNA replication. (g), G mRNA synthesis. (h), N mRNA synthesis. All RNA data were quantified by real-time RT-PCR. All results are from n = 3 independent experiments. Western blots (a), shown are the representatives of n = 3 independent experiments. Folds of G and N protein increases (b and c) are the means of n = 3 biologically independent experiments ± standard deviation. Viral titers (d) and RNA copies (e-h) are the geometric mean titers (GMT) of n = 3 biologically independent experiments ± standard deviation. Statistical significance was determined by two-sided student’s t-test. Exact P values are included in Data Source. *P < 0.05, **P < 0.01, and ***P < 0.001.

Source data

Extended Data Fig. 6 m6A-deficient HMPVs are attenuated in replication in cell culture and are defective in m6A methylation.

(a), Cytopathic effects (CPE) caused by m6A-deficient rHMPV mutants in A549 cells. Confluent A549 cells were infected with each rHMPV at an MOI of 1.0, CPE was imaged at days 2 and 3. All m6A-deficient rHMPV mutants had an earlier CPE compared to rHMPV. (b), Growth curve of m6A-deficient rHMPV mutants in A549 cells. Confluent A549 cells were infected with each rHMPV at an MOI of 1.0, total virus in supernatant and cell lysate was harvested at the indicated time, and viral titer was determined by an immunostaining plaque assay. (c), Immunoblot analysis of HMPV proteins. Confluent A549 cells were infected with each rHMPV at an MOI of 1.0, cell lysates were harvested at 24, 48, and 72 h post-infection, and HMPV proteins were detected by specific antibodies against N and G protein by Western blot. (d), m6A-mutated HMPV RNA 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 Magna MeRIPTM m6A kit. Anti-m6A antibody was first conjugated to magnetic beads. Total RNA (15 µg) was extracted from rHMPV or m6A deficient rHMPV-infected A549 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 was quantified by real-time RT-PCR using primers annealing to HMPV antigenome, genome, and G mRNA. All results are from n = 3 biologically independent experiments. CPE images (a) and Western blots (c) shown are the representatives of n = 3 independent experiments. Viral titers (b) are the geometric mean titers (GMT) of n = 3 independent experiments ± standard deviation. The m6A levels (c) are means of n = 3 independent experiments ± standard deviation. Statistical significance was determined by two-sided student’s t-test. Exact P values are included in Data Source. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Source data

Extended Data Fig. 7 m6A deficient rHMPV trigger a higher type I IFN secretion.

(a), Dynamics of IFN-α secretion in A549 cells infected by HMPV at MOI of 4.0. A549 cells were infected with rHMPV or each rHMPV mutant at an MOI of 4.0, cell culture supernatants were harvested at 16, 24, and 40 h post-inoculation, and IFN-α in cell supernatants were measured by ELISA. A standard curve was generated using human IFN-α. (b), Dynamics of IFN-β secretion in THP-1 cells infected by HMPV at MOI of 1.0. THP-1 cells were infected each HMPV at an MOI of 1.0, and IFN-β in cell culture supernatants harvested at 16, 24, 40, and 48 h post-inoculation was detected by ELISA kit. A standard curve was generated using human IFN- β. (c), Comparison of IFN response of virion RNA of HMPV mutants. Virion RNA was extracted from purified HMPV virions, the level of antigenome was quantified by real-time RT-PCR. A549 cells in 24-well plates were transfected with 1.0 × 105 RNA copies of virion RNA of rHMPV-G1-14, G1-2, G8-9, and rHMPV, and dynamics of IFN response was detected by ELISA kit. (d), Natural m6A-deficient virion RNA induces IFN response. A549 cells were transfected with 1.0 × 105 RNA copies of virion RNA of rHMPV-G1-14,rHMPV-G(-)1–6, rHMPV-ALKBH5, and rHMPV, and dynamics of IFN response was detected by ELISA kit. Data (a-d) shown are means of n = 3 biologically independent experiments ± standard deviation. Statistical significance was determined by two-sided student’s t-test. Exact P values are included in Data Source. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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Extended Data Fig. 8 m6A-deficient rHMPVs and virion RNA induce higher phosphorylation of IRF3.

(a), Removal of 5’ triphosphate abolished RIG-I expression and phosphorylation of IRF3. A549 cells were transfected with virion RNA of rHMPV-G1-14, G8-14, and rHMPV with or without CIP treatment. At 24 h post-transfection, RIG-I was detected by Western blot using antibody against RIG-I. IRF3 phosphorylation was detected by Western blot using antibody specific to IRF3 or phosphorylated IRF3 on site S386 or S396. (b), m6A-deficient rHMPVs induce higher RIG-I expression and IRF3 phosphorylation. A549 cells were infected by each HMPV at an MOI of 5.0. At indicated time points, RIG-I expression and IRF3 phosphorylation at sites S386 and S396 were detected by Western blot. (c), m6A-deficient rHMPVs induce higher phosphorylation of IRF3. Confluent A549 cells were infected by of each HMPV at an MOI of 5.0. At 8, 16, 24, and 32 h post-transfection, cell lysates were subjected to Western blot using antibody specific to IRF3 or phosphorylated IRF3 on site S386 or S396. (d), m6A-deficient virion RNA induce higher phosphorylation of IRF3. Confluent A549 cells were transfected with poly (I:C) or virion RNA from rHMPV, rHMPV-G8-14, and rHMPV-G1-14. At 8, 16, 24, and 32 h post-transfection, cell lysates were subjected to Western blot using antibody specific to IRF3 or phosphorylated IRF3 on site S386 or S396. All Western blots (a-d) shown are the representatives of n = 3 biologically independent experiments.

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Extended Data Fig. 9 Cytopathic effects (CPE) produced by m6A-deficient HMPVs in parental and knockout A549 cells.

Confluent wild-type, MDA5, RIG-I, or MAVs-knockout A549-Dual cells in 24-well plates were infected by rHMPV, rHMPV-G8-14, or rHMPV-G1-14 at an MOI of 5.0. CPE was imaged at 16 h post-infection. Both rHMPV-G8-14 and rHMPV-G1-14 exhibited an earlier CPE compared to rHMPV. Images shown are the representatives of n = 3 biologically independent experiments.

Extended Data Fig. 10 Contribution of other functions of m6A methylation to the attenuated phenotype of m6A-deficient rHMPVs.

G protein expression increased in A549 cells that transiently overexpress m6A reader proteins (a) and writer proteins (c). Conversely, G protein expression was reduced when individual, endogenous reader (YTHDF1-3 and YTHDC1) (b) and writer (METTL3 and METTL14) (d) were knocked down by siRNA. (a) Transient expression of m6A reader proteins enhances G expression. A549 cells were transfected with 1 µg of plasmids encoding YTHDF1, 2, 3, or YTHDC1. At 24 h post-transfection, cells were further transfected with 1 µg of pCAGGS-G-HA. At 24 h post-transfection, total cell extracts were harvested and subjected to Western blot using antibody against HA-tag. (b), siRNA knockdown of m6A reader proteins reduces G expression. A549 cells were transfected with siRNA targeting YTHDF1, 2, 3, or YTHDC1. At 24 h post-transfection, cells were further transfected with 1 µg of pCAGGS-G-HA. At 24 h post-transfection, cell lysates were harvested for Western blot analysis. (c), Transient expression of m6A writer proteins enhances G expression. A549 cells were transfected with 1 µg of plasmids encoding METTL3 and/or METTL14. At 24 h post-transfection, cells were further transfected with 1 µg of pCAGGS-G-HA. At 24 h post-transfection, total cell extracts were harvested and subjected to Western blot using antibody against HMPV G protein. (d), siRNA knockdown of m6A writer proteins reduces G expression. A549 cells were transfected with siRNA targeting METTL3 and/or METTL14. At 24 h post-transfection, cells were further transfected with 1 µg of pCAGGS-G-HA. At 24 h post-transfection, cell lysates were harvested for Western blot analysis. (e), Mutations in m6A sites in G reduces G expression. The m6A sites in the G mRNA were mutated and the G protein expression was determined. A549 cells were transfected with 1 µg of each plasmid. At 24 h post-transfection, cell lysates were harvested for Western blot analysis. Mutants pCAGGS-G1-14 and pCAGGS-G8-14 had a significant reduction in G protein expression compared to pCAGGS-G. Thus, abrogation of m6A site in G mRNA diminished G protein translation. All Western blots (a-e) shown are the representatives of n = 3 biologically independent experiments.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–11, Supplementary Tables 1 and 5, discussion and references.

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Supplementary Tables 2–4.

Supplementary Data 1

Source_Data_Supplementary_Fig. 1. PDF includes unprocessed gel.

Supplementary Data 2

Source_Data_Supplementary_Fig. 1. xls includes statistical source data.

Supplementary Data 3

Source_Data_Supplementary_Fig. 8. PDF includes unprocessed western blots.

Supplementary Data 4

Source_Data_Supplementary_Fig. 8. xls includes statistical source data.

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Source Data Fig. 1

Source_Data_Fig. 1.PDF includes unprocessed western blots.

Source Data Fig. 1

Source_Data_Fig. 1.xls includes statistical source data.

Source Data Fig. 2

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Source_Data_Fig. 3.xls includes statistical source data.

Source Data Fig. 4

Source_Data_Fig. 4.PDF includes unprocessed western blots.

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Source Data Fig. 5

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Source Data Extended Data Fig. 1

Source_Data_ED_Fig. 1.PDF includes unprocessed western blots and gels.

Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 3

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Source Data Extended Data Fig. 5

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Source Data Extended Data Fig. 6

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Lu, M., Zhang, Z., Xue, M. et al. N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I. Nat Microbiol 5, 584–598 (2020). https://doi.org/10.1038/s41564-019-0653-9

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