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.

(a) Distribution of m⁶A peaks in the hMPV antigenome and genome. A schematic diagram of the hMPV antigenome encoding 8 genes is shown. Total RNAs were extracted from purified rhMPV virions grown in A549 cells and were subjected to m⁶A immunoprecipitation followed by m⁶A-seq. Top panel: The m⁶A-seq of hMPV RNA showing the distribution of m⁶A-IP reads (blue block) mapped to the hMPV antigenome. The baseline signal from input samples is shown as a blue line. Lower panel: The distribution of m⁶A-IP reads (pink block) from m⁶A-seq mapped to the hMPV genome. The baseline signal from input samples is shown as a pink line. The red arrows indicate the m⁶A peaks. (b) Distribution of m⁶A peaks in the hMPV mRNAs. Polyadenylated mRNAs were isolated from hMPV-infected cells and subjected to m⁶A-seq. m⁶A reader (c) and writer (g) proteins enhance hMPV protein expression. A549 cells were transfected with plasmids encoding reader (c) or writer (g) genes. At 24 h, cells were infected with rhMPV at an MOI of 5.0. Total cell extracts were analyzed by Western blot. m⁶A reader (d) and writer (f) proteins increase hMPV progeny virus production. The release of infectious hMPV particles was monitored by a single-step growth curve. (e) YTHDF1, 2, 3 enhance GFP expression in rghMPV-infected cells. HeLa cells stably overexpressing YTHDF proteins were infected with rghMPV at an MOI of 1.0, and GFP expression was monitored at 48 h post-infection. (h) Strong co-localization of METTL14 with hMPV N protein. A549 cells were infected by rhMPV at an MOI of 5.0. At 24 h, cells were stained with anti-METTL14 antibody (green) and anti-hMPV N antibody (red), and analyzed by confocal microscope. Nuclei were labeled with DAPI (blue). The results of n = 2 (a-b), n = 3 (c, e-h), or n = 4 (d) biologically independent experiments are shown (representative immunoblots (c, g) and images (e, h) are shown). Data (a and b) are the average results from two samples (n = 2). Viral titers are the geometric mean titers (GMT) ± 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.

Fig. 2.. m⁶A-deficient rhMPVs and their virion RNAs induce higher type I IFN responses.

(a) Immunostaining spots formed by rhMPVs. (b) Quantification of m⁶A level. Total m⁶A level of each virion RNA was quantified by m⁶A RNA Methylation Assay Kit. (c) m⁶A-deficient rhMPV RNA has reduced binding efficiency to reader proteins. Cell lysate containing HA-tagged YTHDF1 or YTHDF2 was incubated with virion RNA and anti-HA Magnetic beads. The amount of virion RNA captured by the YTHDF1 or YTHDF2 was quantified by real-time RT-PCR. Percent of bound RNA of hMPV mutants relative to rhMPV was calculated. IFN-β secretion in A549 cells infected by hMPV at MOI of 4.0 (d, h) or an MOI of 1.0 (e). A549 cells were infected with each rhMPV at an MOI of 4.0, and IFN-β in cell supernatants were measured by ELISA. Dynamics of IFN-β secretion in THP-1 cells infected by hMPV at an MOI of 4.0 (f-g). (i) IFN-β response in A549 cells transfected with total RNA. Total RNA was extracted from hMPV-infected A549 cells, and the antigenome was quantified by real-time RT-PCR. A549 cells were transfected with 10⁸ antigenome RNA copies of total RNA with or without treatment CIP. IFN-β was measured by ELISA. (j) IFN-β response in A549 cells transfected with viral G mRNA. A549 cells were transfected with 10⁹ RNA copies of G mRNA either with or without CIP treatment. (k) IFN-β response in A549 cells transfected with virion RNA. A549 cells were transfected with 2×10⁷ antigenome copies of virion RNA either with or without CIP treatment. (l and m) Comparison of IFN response of virion RNA of rhMPV-G1-14, G1-2, G8-9, and rhMPV. A549 cells were transfected with 10⁷ (l) or 10⁶ (m) RNA copies of virion RNA. (n and o) Natural m⁶A-deficient virion RNA induces IFN response. A549 cells were transfected with 10⁷ (n) or 10⁶ (o) RNA copies of virion RNA of rhMPV-G1-14, G(-)1-6, ALKBH5, and rhMPV. Immunospots (a) shown are the representatives of n = 3 biologically independent experiments. Data shown are means of n = 3 (c-o) or n = 4 (b) 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; ****P<0.0001, NS, no significant.

Fig. 3.. IFN response and NF-κB activation in A549 cells infected with m⁶A deficient hMPVs or transfected with m⁶A deficient virion RNA.

Confluent WT (a), MDA5 (b), RIG-I (c), or MAVS (d)-knockout A549 cells were infected by rhMPV, rhMPV-G8-14, or rhMPV-G1-14 at an MOI of 1.0, cell culture supernatants were harvested at 24 and 48 h post-inoculation. IFN-β in cell supernatants was measured by ELISA. Confluent wild-type (e), MDA5 (f), RIG-I (g), or MAVs (h)-knockout A549 cells were transfected with 10⁷ antigenomic RNA copies of virion RNA of rhMPV, rhMPV-G8-14, rhMPV-G1-14 or 2 μg poly(I:C), cell culture supernatants were harvested at 24 and 40 h post-inoculation. IFN-β in cell supernatants was measured by ELISA. Confluent WT (i), MDA5 (j), RIG-I (k), or MAVs (l)-knockout A549 cells were infected by rhMPV, rhMPV-G8-14, or rhMPV-G1-14 at an MOI of 1.0, cell culture supernatants were harvested at 24 and 48 h post-inoculation. These A549 cells also express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB binding sites, allowing us to measure the activation of the NF-κB pathway. SEAP secreted in cell supernatants was measured by colorimetric enzyme assay with substrate Quanti-Blue™ and read by microplate reader on OD value at 620nm. Confluent WT (m), MDA5 (n), RIG-I (o), or MAVs (p)-knockout A549 cells were transfected with 10⁷ antigenomic RNA copies of virion RNA of rhMPV, rhMPV-G8-14, rhMPVG1-14 or 2 μg poly(I:C) with or without CIP treatment, cell culture supernatants were harvested at 16, 24, and 40 h post-inoculation. SEAP secretion in cell supernatants was measured. Data shown (a-l) are means of n =3 or n = 4 (m-p) biologically independent experiments ± standard deviation. All data of rhMPV-G8-14 and rhMPV-G1-14 were compared to those of rhMPV. 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; ****P<0.0001.

Fig. 4.. m⁶A-deficient hMPVs and virion RNA induce a higher expression of RIG-I.

(a) m⁶A-deficient rhMPVs stimulate a higher expression of RIG-I. A549 cells were infected by each hMPV at an MOI of 0.2, 1.0, and 5.0. At indicated times, cell lysates were subjected to Western blot analyses using antibody specific to RIG-I, hMPV N, or β-actin. (b) m⁶A-deficient virion RNA induces a higher expression of RIG-I. A549 cells were transfected with an increasing amount of poly (I:C) (0.5 and 2.0 µg/well) or virion RNAs (2×10⁵, 2×10⁶, or 2×10⁷ copies/well) of rhMPV, rhMPV-G8-14, or rhMPV-G1-14. At indicated times, cell lysates were subjected to Western blot analysis using antibody against RIG-I or β-actin. (c) Comparison of RIG-I expression triggered by virion RNA. A549 cells were transfected with increasing amounts (10⁵, 10⁶, and 10⁷ RNA copies) of virion RNA of rhMPV, rhMPV-G1-2, rhMPV-G8-9, or rhMPV-G1-14. (d) Removal of 5’ triphosphate abolished RIG-I expression and IRF3 phosphorylation. A549 cells were transfected with virion RNA with or without CIP treatment. At indicated times, cell lysates were subjected to Western blot using antibody specific to IRF3 or phosphorylated IRF3 (pIRF3) on site S386 or S396. (e) Natural m⁶A-deficient rhMPVs induce higher phosphorylation of IRF3. A549 cells were infected by each hMPV at an MOI of 5.0. At indicated times, RIG-I expression and IRF3 phosphorylation was detected by Western blot. (f) Natural m⁶A-deficient virion RNA induces higher RIG-I expression. 10⁷ copies of virion RNA were used for transfection. Western blots (a-f) shown are the representatives of n =3 biologically independent experiments. (g) Model for RIG-I mediated IFN signaling pathway. Upon hMPV entry, the RNP complex is delivered into the cytoplasm where RNA synthesis and viral replication occur. During replication, the RdRP initiates at the extreme 3’ end of the genome and synthesizes a full-length complementary antigenome, which subsequently serves as template for synthesis of full-length progeny genomes. The newly synthesized genome and antigenome was methylated by m⁶A writer proteins and encapsidated by viral N protein. Viral genome and antigenome are recognized by cytoplasmic RNA sensor RIG-I and induces signaling to the downstream adaptor protein MAVS which subsequently activates IRF3 and NF-κB pathways, leading to the production of type-I IFN. The internal m⁶A methylation on virion RNA inhibits RIG-I mediated IFN signaling pathway.

Figure 5.. m⁶A-deficient virion RNA increases RIG-I binding affinity and facilitates RIG-I:RNA conformation change.

(a) Biotinylated virion RNA pulldown RIG-I. Biotinylated virion RNA was conjugated to Streptavidin beads and incubated with A549 cell lysate containing overexpressed RIG-I. The pull-down RIG-I protein was detected by Western blot. (b and c) RIG-I pulldown hMPV RNA. RIG-I conjugated magnetic beads were incubated with virion RNA, N or G mRNA. One aliquot of beads was subjected for Western blot (b). RNA bound to magnetic beads was quantified by real-time RT-PCR (c). (d) Purified Flag-tagged RIG-I protein. (e) Competitive binding of WT virion RNA and m⁶A-deficient virion RNA to RIG-I. Streptavidin beads-bound rhMPV-G1-14 and rhMPV RNA were mixed at different ratios and incubated with RIG-I protein in the presence of AMP-PNP. RIG-I pulldown was detected by Western blot. (f) Domain structure of RIG-I protein. CARD, caspase activation and recruitment domains; Helicase, helicase domain; CTD, C-terminal domain. Red flashes indicate trypsin cleavage sites. (g) Model for mechanisms of enhanced RIG-I-mediated IFN signaling by m⁶A-deficient hMPV RNA. RIG-I is in an autorepressed conformation in the absence of ligand. RIG-I CTD recognizes and binds to 5’triphosphate of RNA. 5’triphosphate RNA without m⁶A has a higher binding affinity to helicase domain of RIG-I. RIG-I is an RNA translocase, moving from 5’-ppp to RNA chain. Internal m⁶A may serve as a “brake” to prevent RIG-I translocation (indicated by question mark). The RIG-I helicase domain binds the RNA, triggering RIG-I conformational change and subsequent oligomerization. RNAs without m⁶A more easily induce RIG-I conformational change. The released CARDs of the activated RIG-I:RNA complex are ubiquitinated for downstream signaling. (h-k) Analysis of RIG-I:RNA conformation by limited trypsin digestion. Limited trypsin digestion of RIG-I protein in the absence of RNA ligand for 0–2 h (h), or in the presence of poly (I:C) (i) or virion RNA (j) for 2h was shown. (k) Competition assay. RIG-I incubated with mixtures containing different ratios of RNA of rhMPV-G1-14 and rhMPV, and digested by trypsin for 2h. RIG-I fragments were detected by Western blot. Black arrows in h–k indicate the 80 kDa trypsin-resistant protein band. Western blots (a, b, e, h-k) and SDS-PAGE (d) shown are the representatives of n = 3 biologically independent experiments. Data shown (c) are means of n =3 independent experiments ± standard deviation. Statistical significance was determined by two-sided student’s t-test. *P<0.05; **P<0.01; ***P<0.001.

Fig. 6.. Replication, interferon response, pathogenicity, and immunogenicity of m⁶A-deficient rhMPVs. Replication Kinetics of m⁶A deficient rhMPVs in WT

(a), MDA5 (b), RIG-I (c), or MAVS (d)-knockout A549 cells. Cells in 24-well plates were infected by each hMPV at an MOI of 1.0, and viral growth curve was determined. The arrows indicate the degree of titer difference compared to rhMPV. (e) Interferon response of rhMPV in cotton rats. Six-week-old SPF female cotton rats (n = 5) were inoculated intranasally with 100 µl of PBS or 2.0×10⁵ p.f.u. of rhMPV-G8-14, rhMPV-G1-14 or rhMPV. At 48 h post-inoculation, cotton rats were sacrificed. BAL from the right lung was collected for IFN-β bioactivity assay. (f) hMPV titer in lungs and nasal turbinates. Six-week-old SPF cotton rats (n = 5) were inoculated intranasally with 2.0×10⁵ p.f.u. of each rhMPV. At day 4 post-infection, lungs and nasal turbinates were collected for virus titration. (g) m⁶A deficient rhMPVs had less lung histopathological changes compared to rhMPV. Representative pathological changes from each group are shown. Micrographs with original magnification, ×20 are shown. The parental hMPV caused moderate interstitial pneumonia, mononuclear cell infiltration. In contrast, fewer histological changes were found in the lungs of cotton rats infected with m⁶A-deficient rhMPVs. (h) m⁶A deficient rhMPV provides complete protection against hMPV challenge. Four-week-old SPF cotton rats (n = 5) were inoculated intranasally with 2.0×10⁵ p.f.u. of each rhMPV. At week 4 post-immunization, cotton rats were challenged with 2.0×10⁵ p.f.u. of hMPV. At day 4 post-challenge, the cotton rats were sacrificed, lungs and nasal turbinates were collected for virus titration by an immunostaining plaque assay. (i) m⁶A deficient rhMPV induced a high level of neutralizing antibody. Blood samples were collected from each rat weekly by retro-orbital bleeding. The hMPV-neutralizing antibody titer was determined using a plaque reduction neutralization assay. Viral titers (a-d) are the geometric mean titers (GMT) of n = 3 biologically independent experiments ± standard deviation. Viral titers (f and h) and antibody titers (i) are the geometric mean titers (GMT) of five cotton rats (n = 5) ± standard deviation. Detection limit is 2.0 log [p.f.u.] per g tissue. 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; ****P<0.0001.