The animal study was conducted in strict accordance with USDA regulations and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council and was approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC; animal protocol no. 2009A0221). The animals were housed within the University Laboratory Animal Resources (ULAR) facilities of The Ohio State University according to the guidelines of the Institutional Animal Care and Use Committee (IACUC). The animal care facilities at The Ohio State University are AAALAC accredited. Every effort was made to minimize potential distress, pain, or discomfort to the animals throughout all experiments.
HeLa (ATCC CCL-2), A549 (ATCC CCL-185), Vero (ATCC CRL-CCL81), and HEp-2 (ATCC CCL-23) cell lines were purchased from the American Type Culture Collection (Manassas, VA) and were grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% FBS. HeLa cells overexpressing the empty vector (pPB-CAG), YTHDF1, YTHDF2, or YTHDF3 were maintained in DMEM, 10% FBS and 1 µg/ml of puromycin every passage to select for YTHDF1-3 overexpressing cells. Primary, well-differentiated human airway epithelial (HAE) cultures were grown on collagen coated Transwell inserts (Corning Incorporated, Corning, NY) at an air-liquid interface, as previously described47. Upon reaching confluency and forming tight junctions, the apical medium was removed and cultures were maintained at the air–liquid interface for 4–6 weeks to generate well-differentiated, polarized cultures. All cell lines used in this study were free of mycoplasma, as confirmed by the LookOut Mycoplasma PCR Detection Kit (Sigma).
Recombinant RSV containing a green fluorescence protein (GFP) gene between the leader sequence and NS1 gene (rgRSV)47 was propagated and titered in HeLa cells or A549 cells. To prepare purified rgRSV, 20 T150 flasks of HeLa cells or A549 cells were infected by rgRSV at an MOI of 0.1, and cell culture supernatants harvested at 48 or 72 h post-infection were clarified by centrifugation at 10,000 × g for 30 min. Virus was concentrated through a 35% (wt/vol) sucrose cushion by centrifugation at 30,000 × g for 2 h at 4 °C in a Ty 50.2 rotor (Beckman). The pellet was resuspended in DMEM with 10% trehalose and further purified through a sucrose gradient (20–55%) by centrifugation at 35,000 × g for 2 h at 4 °C in an SW55 rotor (Beckman). The final pellet was resuspended in 0.5 ml of DMEM with 10% trehalose.
High-throughput sequencing of the RSV and host methylome was carried out using m6A-seq (MeRIP-seq) as described previously20. For m6A-seq of the rgRSV genome and antigenome, RNAs were extracted from purified rgRSV virions and purified with the RiboMinus Eukaryote System v2 kit (Thermo Fisher). For m6A-seq of host transcripts, total RNAs were extracted from mock or rgRSV-infected HeLa or A549 cells and polyadenylated RNAs were isolated using Dynabeads mRNA DIRECT Purification kit (Thermo Fisher). Purified RNAs were sonicated with Bioruptor Pico (Diagenode) with 30s ON 30s OFF for 30 cycles, mixed with 1 µl of affinity purified anti-m6A monoclonal antibody (NEB) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) and incubated for 2 h at 4°C. Enriched mRNA fragments were purified with RNA Clean & Concentrator kit (Zymo) and used for library generation with TruSeq Stranded mRNA Library Prep kit (Illumina). Sequencing was carried out on Illumina HiSeq 4000 according to the manufacturer’s instructions. Two replicates of RNA samples from virions, virus-infected cells, and mock-infected cells were subjected to m6A-seq. For data analysis, after removing the adapter sequences, the reads were mapped to the human genome (hg38) and rgRSV genome and antigenome by using Hisat256 with peak calling as described57. Metagene analysis was performed by R package Guitar58. Differential methylation analysis was performed with count based negative binomial model implemented in QNB test46.
RSV RNA (250 mg) was extracted from highly purified rgRSV virions using an RNeasy Mini kit (Qiagen) and purified twice with RiboMinus Eukaryote System v2 kit (Thermo Fisher). To examine the purity of virion RNA, oligo d(T) was used for reverse transcription, followed by qPCR for quantification for β-actin and viral N and G mRNAs. Virion RNA which was free of contamination of host RNA and viral mRNAs was used for liquid chromatography-mass spectrometry (LC-MS/MS), m6A antibody pulldown assay, and m6A-seq. Purified RNA was digested and subjected to a quantitative analysis of the m6A level using LC-MS/MS as previously described7.
A549 cells in T150 flasks were transfected with 10 µg of plasmids encoding METTL3 and METTL14, ALKBH5, or vector pCAGGS. For siRNA transfection, A549 cells in T75 flasks were transfected with 150 pmole of siRNA targeting METTL3 and METTL14, ALKBH5, or control siRNA. At 24 h post-transfection, the transfected cells were infected with rgRSV at an MOI of 0.1. At 42 h post-infection, cell culture supernatants (containing RSV particles) were harvested. RSV particles were pelleted and purified through ultracentrifugation. Virion RNA was extracted from highly purified RSV virions. Antigenome was quantified by real-time RT-PCR. Each amount of antigenome was bound to strip wells using a RNA high binding solution, and m6A was detected using a specific capture anti-m6A antibody (Abcam, ab185912) and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. A standard curve was generated using known m6A methylated RNA (range from 0.02 to 1 ng of m6A) as a positive control. The m6A content was calculated from each RNA samples based on their OD450 values. The percent of changes was calculated by dividing m6A contents in viral RNA from the treated group by those from the control group.
Host cell differential gene expression was analyzed by R package DESeq258 using wald-test. The significantly differentially expressed genes were reported at adjusted P value cutoff of 0.05.
Gene Ontogeny (GO) analysis was performed using the R package cluster Profiler58. Specifically, enrichKEGG function was called to analyze for enriched pathway and enrichMap function was called to generate network plot of enriched pathway.
The plasmid vector was used to overexpress the readers (YTHDF1-3), writers (METTL3, METTL14), and erasers (FTO, ALKBH5) as described previously32. Plasmid (RW30) encoding the full-length antigenomic cDNA of RSV strain A2 with GFP inserted between the leader and the NS1 gene, and support plasmids expressing RSV A2 strain N protein (pTM1-N), P protein (pTM1-P), L protein (pTM1-L), and M2-1 protein (pTM1-M2-1) were generously provided by Dr. P.L. Collins, NIAID, Bethesda, MD. Mutations to the potential m6A sites in G gene were introduced into the RW30 plasmids using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The m6A peaks in the G gene are clustered in three regions, 392–467 nt, 567–660 nt, and 716–795 nt. Since it is known that m6A modified sites in RNA contain the conserved Pu [G > A]m6AC[A/C/U] motif (Pu represents purine)1, we searched for this motif in these three regions in G mRNA and identified 6, 7, and 4 potential m6A sites in regions G1, G2, and G3 respectively (Supplementary Fig. 13). The potential m6A sites mutants in G1 peak include 394-AGm6ACC-400; 401-AAm6ACA-407; 418-AAm6ACA-424; 444-AAm6ACA-450; 455-AAm6ACA-461; 459-AAm6ACC-465; mutants in G2 peak include 569-AAm6ACA-575; 576-AAm6ACC-582; 589-AAm6ACC-595; 612-AAm6ACC-618; 625-AGm6ACA-631; 645-AAm6ACC-651; 652-AAm6ACC-658; and mutants in G3 peak include 718-AAm6ACA-724; 722-AAm6ACA-728; 768-GAm6ACT-774; 787-AAm6ACC-793 (Supplementary Fig. 13). The A or C within the consensus m6A sites was mutated to a T or G in these sites without changing the encoded amino acid. Mutant G12 combined the mutations from G1 and G2. Mutant G123 was a combined the mutations from G1, G2, and G3. In addition, M-fold and Genscript software were used to predict that these mutations did not alter RNA secondary structure or codon usage. All plasmids and mutations were confirmed by DNA sequencing.
siRNAs against METTL3, METTL14, FTO, ALKBH5, YTHDF1, YTHDF2, YTHDF3, or non-targeting AllStars negative control siRNA were purchased from Qiagen (Valencia, CA, sequences listed in Supplementary Table 7). All siRNA transfections were performed using the Lipofectamine 3000 transfection reagent (Thermo-Fisher) according to the manufacturer’s instructions.
The antibodies used in this study were anti-YTHDF1 (1:1000, Proteintech, Rosemont, IL), anti-YTHDF2 (1:1000, Abcam, Cambridge, MA), anti-YTHDF3 (1:1000, Abcam), anti-METTL3 (1:1000, Proteintech), anti-METTL14 (1:1000, Proteintech), anti-ALKBH5 (1:1000, Sigma-Aldrich, St. Louis, MO), anti-FTO (1:1000, Abcam), and anti-RSV serum (1:400, Virostat, Westbrook, ME), F (1:3000, Abcam), anti-FLAG (1:3000, Sigma-Aldrich), anti-Actin (1:5000, Proteintech), and anti-Tubulin (1:5000, Abcam). Cells were harvested and lysed in RIPA buffer (Abcam) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Western blotting was performed as described. Tubulin or actin was used as a loading control.
Mock or rgRSV-infected cells were fixed in acetone and methanol at the ratio of 1:1 for 30 min, and blocked with 10% goat serum. Slides were stained with all primary antibodies (1:100), washed three times with PBST, and stained with conjugated Alexa Fluor secondary antibodies Alexa Fluor 488/594 (1:300, Thermo-Fisher), and mounted with SlowFade™ Diamond Antifade Mountant with DAPI (Thermo-Fisher). Imaging was performed on an Olympus FV 1000 confocal microscopy system at The Ohio State University Campus Microscopy & Imaging Facility.
RSV genome, antigenome, and mRNA were quantified by real-time RT-PCR. HeLa or A549 cells were infected with rgRSV or an rgRSV mutant at an MOI of 0.1. At 12, 18, and 24 post-infection, total RNA was isolated from cells using TRIzol (Life Technologies). Viral genome or antigenome copies were quantified by real-time RT-PCR using two primers specifically targeting the RSV leader sequence and GFP gene (Supplementary Table 8). Poly (A)-containing viral mRNAs were isolated from total RNA using a Dynabead mRNA isolation kit (Life Technologies) according to the manufacturer’s recommendations. Using the viral mRNAs as the template, the NS1 and G mRNA copies were quantified by real-time RT-PCR using two primers targeting the viral NS1 and G genes, respectively.
The RNA-immunoprecipitation (RIP) assay was performed as described previously38. In brief, HeLa cells were infected with rgRSV at MOI of 1.0 and cell extracts were harvested in polysome lysis buffer after 36 h post-infection. RNP complexes were immunoprecipitated with anti-HA antibody conjugated to magnetic beads (Sigma) or anti-YTHDF2 antibody overnight at 4 °C, and washed five times with ice-cold NT2 buffer. For the RIP with anti-YTHDF2 antibody, additional secondary antibody was added. After the final wash, 10% of the beads were used for immunoblotting and the remaining 90% were used for RNA extraction using TRIzol (ThermoFisher).
rgRSV mutants were rescued from the full-length cDNA of the RSV A2 strain59. HEp-2 cells were infected with MVA-T7 at an MOI of 10, then transfected with 1.2 µg of plasmid RW30 or RW30 mutant, 0.4 µg of pTM1-N, 0.2 µg of pTM1-P, 0.1 µg of pTM1-M2-1, and 0.1 µg of pTM1-L using the Lipofectamine 3000 reagent (Life Technologies). At day 4 post-transfection, the cells were harvested using scrapers and were co-cultured with new flask of HEp-2 cells at 50–60% confluence. When an extensive cytopathic effect (CPE) was observed, the cells were subjected to three freeze-thaw cycles, followed by centrifugation at 4000 × g for 10 min. The supernatant was subsequently used to infect new HEp-2 cells. The successful recovery of the rgRSV was confirmed by the presence of green fluorescent cells, followed by RT-PCR and sequencing. Recombinants rgRSV carrying mutations in m6A sites were designated as rgRSV-G1, G2, G3, G12, and G123.
All plasmids, viral mutants and stocks, and virus isolates from the nasal turbinates and lungs of cotton rats were sequenced to confirm virus identity. Viral RNA was extracted from 100 µl of each recombinant virus using an RNeasy minikit (Qiagen, Valencia, CA). A 1.5-kb DNA fragment spanning the RSV G gene was amplified by RT-PCR. The PCR products were purified and sequenced using a sequencing primer at The Ohio State University Plant Microbe Genetics Facility to confirm the presence of the designed mutations.
Confluent HeLa or A549 cells in 6-well-plate were infected with wild-type rgRSV or mutant rgRSV at an MOI of 0.1. After 1 h of adsorption, the inoculum was removed and the cells were washed three times with DMEM. Fresh DMEM (supplemented with 2% FBS) was added, and the infected cells were incubated at 37 °C. At different time points post-inoculation, the supernatant and cells were harvested by three freeze-thaw cycles, followed by centrifugation at 1500 × g at room temperature for 15 min. The virus titer was determined by TCID50 assay in HEp-2 cells47.
Confluent Vero cells in T25 flasks were infected with each rgRSV mutant at an MOI of 0.1. At day 3 post-inoculation, the cell culture supernatant was harvested and used for the next passage in Vero cells. Using this method, each rgRSV mutant was repeatedly passaged 15 times in Vero cells. At each passage, the G gene was amplified by RT-PCR and sequenced. At passage 15, the entire genome of each recombinant virus was amplified by RT-PCR and sequenced.
Thirty 6-week-old specific-pathogen-free (SPF) male cotton rats (Envigo, Indianapolis, IN) were randomly divided into 6 groups (5 cotton rats per group). Prior to virus inoculation, the cotton rats were anesthetized with isoflurane. The cotton rats in group 1 were inoculated with 2.0 × 105 TCID50 of parental rgRSV and served as positive controls. The cotton rats in groups 2–5 were inoculated with 2.0 × 105 TCID50 of four m6A deficient rgRSV mutants, rgRSV-G1, G2, G3, and G12. Each cotton rat was inoculated intranasally with a volume of 100 μl. At day 4 post-infection, the cotton rats were killed via carbon dioxide inhalation. The left lung and nasal turbinates were collected for virus titration and the right lung was collected for histological analysis.
For the immunogenicity study, twenty 6-week-old female cotton rats (Envigo) were randomly divided into five groups (5 cotton rats per group). Cotton rats in groups 1, 2, and 3 were intranasally inoculated with 2.0 × 105 TCID50 of two m6A deficient rgRSV mutants (rgRSV-G1 and G12) and rgRSV, respectively. Cotton rats in groups 4 were mock-infected with DMEM and served as unvaccinated challenged control. After immunization, the cotton rats were evaluated daily for any possible abnormal reaction and blood samples were collected from each cotton rat weekly by facial vein retro-orbital plexus sampling, and serum was used for detection of neutralizing antibodies. At 4 weeks post-immunization, the cotton rats in groups 2–5 were challenged with 2.0 × 105 TCID50 of parental rgRSV via intranasal route, and evaluated twice daily for the presence of any clinical symptoms. At 4 days post-challenge, all cotton rats were euthanized by CO2 asphyxiation, and their lungs and nasal turbinates were collected for virus titration. The immunogenicity of rgRSV mutants was assessed based on their ability to trigger neutralizing antibody, the ability to prevent rgRSV replication in lungs and nose, and the ability to protect lung from pathological changes.
After killing, the right lung of each animal was removed, inflated, and fixed with 4% neutral buffered formaldehyde. Fixed tissues were embedded in paraffin and a microtome used to generate 5 μm sections. Slides were then stained with hematoxylin-eosin (H&E) for the examination of histological changes by light microscopy. Histopathological changes were evaluated based on the extent of interstitial inflammation, edema, and peribronchiolar inflammation.
The nasal turbinate and the left lung from each cotton rat were removed, weighed, and homogenized in either 3 ml or 2 ml of DMEM. The lung was homogenized using a Precellys 24 tissue homogenizer (Bertin, MD) by following the manufacturer’s recommendations. The nasal turbinates were homogenized by hand with a 15-mL capacity PYREX® homogenizer (Corning, NY). The presence of infectious virus was determined by TCID50 assay in HEp-2 cells.
RSV-specific neutralizing antibody titers were determined using a plaque reduction neutralization assay. In brief, cotton rat sera were collected by retro-orbital plexus sampling weekly until challenge. The serum samples were heat inactivated at 56°C for 30 min. Twofold dilutions of the serum samples were mixed with an equal volume of DMEM containing ~50 TCID50/well rgRSV in a 96-well plate, and the plate was incubated at room temperature for 1 h with constant rotation. The mixtures were then transferred to confluent HEp-2 cells in a 96-well plate in triplicate. After 1 h of incubation at 37°C, the virus-serum mixtures were removed and the cells were overlaid with 0.75% methylcellulose in overlay media (1 × MEM, 2% FBS, Sodium bicarbonate, 25 mM HEPES, 1% -Glutamine, 1% Pen Strep) and incubated for another 3 days before counting the fluorescent foci. The numbers of foci at each serum dilution were plotted and the 50% plaque reduction titer was used as the RSV-specific neutralizing antibody titer.
Quantitative analysis was performed by either densitometric scanning of autoradiographs or by using a phosphorimager (Typhoon; GE Healthcare, Piscataway, NJ), ImageQuant TL software (GE Healthcare, Piscataway, NJ), and Image J (NIH, Bethesda, MD). Statistical analysis was performed by one-way multiple comparisons using SPSS (version 8.0) statistical analysis software (SPSS Inc., Chicago, IL) or Student’s t-test. A P value of <0.05 was considered statistically significant.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
