Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication

Article

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication Graphical Abstract

Authors David G. Courtney, Edward M. Kennedy, Rebekah E. Dumm, Hal P. Bogerd, Kevin Tsai, Nicholas S. Heaton, Bryan R. Cullen

Correspondence [email protected]

In Brief Influenza A virus (IAV) transcripts bear numerous epitranscriptomic m6A modifications. Courtney et al. map these modifications on both the IAV mRNA and vRNA strands and demonstrate that m6A increases viral RNA expression in cis. Moreover, IAV mutants lacking HA sites on the viral HA segment show reduced pathogenicity in vivo.

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m6A sites on influenza A virus (IAV) mRNAs and vRNAs were mapped High levels of m6A modification increase IAV RNA expression IAV mutants lacking m6A sites on the HA segment are attenuated in culture These same IAV HA m6A mutants show reduced pathogenicity in mice

Courtney et al., 2017, Cell Host & Microbe 22, 377–386 September 13, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.chom.2017.08.004

Cell Host & Microbe

Article Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication David G. Courtney,1,2 Edward M. Kennedy,1,2 Rebekah E. Dumm,1 Hal P. Bogerd,1 Kevin Tsai,1 Nicholas S. Heaton,1 and Bryan R. Cullen1,3,* 1Department

of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, USA authors contributed equally 3Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2017.08.004 2These

SUMMARY

Many viral RNAs are modified by methylation of the N6 position of adenosine (m6A). m6A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m6A-modified RNAs, but the effects of m6A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m6A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m6A ‘‘reader’’ protein YTHDF2 increases IAV gene expression and replication. To address whether m6A residues modulate IAV RNA function in cis, we mapped m6A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m6A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m6A residues in IAV transcripts enhance viral gene expression.

INTRODUCTION The covalent modification of individual bases on mRNA transcripts has recently emerged as a potentially critical mechanism for the post-transcriptional regulation of gene expression (Li and Mason, 2014; Meyer and Jaffrey, 2014). Analysis of the cellular epitranscriptome, defined as internal single nucleotide modifications that do not alter the mRNA sequence, has identified at least ten different modifications, of which the most prevalent is the addition of a methyl group to the N6 position of adenosine, referred to as m6A. It has been reported that the average
2.2 kb cellular mRNA contains three internal m6A residues, and highly regulated mRNAs may contain ten or more m6As (Desrosiers et al., 1975; Linder et al., 2015). Moreover, addition of m6A has been proposed to regulate mRNA function at multiple steps, including splicing, stability, translation, and secondary structure (Li and Mason, 2014; Meyer and Jaffrey, 2014). The cellular machinery that adds m6A to mRNAs and detects its presence is highly conserved in multicellular eukaryotes and

the global loss of m6A results in profoundly deleterious phenotypes, including embryonic lethality, in species ranging from plants to vertebrate animals (Batista et al., 2014; Geula et al., 2015; Hongay and Orr-Weaver, 2011; Zhong et al., 2008). m6A is added co-transcriptionally to nuclear pre-mRNAs by a protein complex consisting minimally of the methyltransferase METTL3 and two co-factors, METTL14 and WTAP, known collectively as m6A ‘‘writers’’ (Ke et al., 2017; Li and Mason, 2014; Meyer and Jaffrey, 2014). Once mRNAs have entered the cytoplasm, they encounter three m6A ‘‘reader’’ proteins called YTHDF1, YTHDF2, and YTHDF3, which are thought to mediate many of the phenotypic effects exerted by m6A. In addition to the important but still emerging role played by m6A in regulating cellular mRNA function, m6A has also been detected on every viral mRNA transcript examined so far including, perhaps unexpectedly, mRNAs encoded by several cytoplasmic RNA viruses (Gonzales-van Horn and Sarnow, 2017; Kennedy et al., 2017). The first virus found to express mRNAs bearing internal m6A residues was influenza A virus (IAV), which was reported to contain
24 m6A residues on the various viral mRNAs, of which the highest number, eight m6As, was detected by biochemical analysis of the hemagglutinin (HA) mRNA segment (Krug et al., 1976; Narayan et al., 1987). However, these m6A residues were not mapped and no examination of how m6A affects IAV gene expression or replication has been reported. Nevertheless, it is known that the drug, 3-deazaadenosine (DAA), which inhibits m6A addition by depleting intracellular levels of the methyl donor S-adenosylmethionine (SAM) (Bader et al., 1978; Fustin et al., 2013) is a potent inhibitor of IAV replication at doses that do not exert any evident cytotoxic effect (Fischer et al., 1990), thus suggesting that m6A plays a positive effect in the IAV life cycle. While few reports as yet exist addressing how m6A affects viral gene expression and replication, this has been examined in the case of HIV-1. Three groups have reported the mapping of multiple m6A residues on the HIV-1 RNA genome and these groups have also examined the effect of overexpressing or knocking down the cellular reader and/or writer proteins on HIV-1 replication (Kennedy et al., 2016; Lichinchi et al., 2016a; Tirumuru et al., 2016). Two groups reported that m6A exerted a positive effect on HIV-1 replication (Kennedy et al., 2016; Lichinchi et al., 2016a), while a third group reported that m6A exerted an inhibitory effect (Tirumuru et al., 2016). As m6A is added to A residues in a well-established sequence consensus (minimally 50 -RAC-30 , where R is purine), it is unclear why HIV-1, which rapidly escapes from other cis-acting inhibitory sequence

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Figure 1. IAV Gene Expression Is Greatly Reduced in METTL3 Knockout Cell Lines (A) Treatment of cells with a non-toxic dose of DAA, an inhibitor of m6A addition, reduced expression of the IAV proteins NS1 and M2 in A549 cells, as determined by western blot. A549 cells were infected at an MOI of 1.0. (B) Two clonal A549 METTL3 knockout cell lines, M3.1 and M3.2, were established using gene editing and confirmed by western blot and genomic sequencing. See Figure S1 for genomic sequences and Table S1 for sgRNA sequences. (C) Two A549 METTL3 knockout lines and two control cell lines, including the parental A549 cell line and a GFP-Flag expressing A549 cell line, were infected with IAV-PR8 at an MOI of 0.01 and NP, NS1, and M2 protein expression determined by western blot at 24, 48, and 72 hr post-infection (hpi). Quantification of the band intensities for NS1 and M2 at 72 hpi revealed expression levels of 0.12 ± 0.01 and 0.12 ± 0.07 for M3.1 and 0.09 ± 0.03 and 0.08 ± 0.03 for M3.2, respectively, when expression in wild-type cells was normalized to 1.0. Actin was used as the loading control. (D) qRT-PCR was performed to determine the levels of the spliced IAV M2 mRNA at the same time points post-infection. See Table S2 for primer sequences. (E) Viral production from the wild-type and METTL3 KO cells at 72 hpi was quantified by plaque assay on MDCK cells. These data represent the average of three biological replicates with SD indicated. *p < 0.05, **p < 0.01.

elements, such as small interfering RNA targets (Boden et al., 2003; Das et al., 2004), should retain m6A sites if these are indeed inhibitory. Another group looked at m6A residues present on the genome of the cytoplasmic RNA virus hepatitis C virus (HCV) and concluded these did not significantly affect viral RNA replication but did reduce the production of progeny viral particles (Gokhale et al., 2016). Similarly, inhibition of m6A addition was also reported to enhance the production of infectious viral particles in cells infected with the cytoplasmic RNA virus Zika virus (ZKV) (Lichinchi et al., 2016b). In this manuscript, we examine how m6A addition affects IAV gene expression and replication. We first demonstrate that mutational inactivation of the key m6A writer METTL3 in the human lung epithelial cell line A549 inhibits IAV replication, while ectopic overexpression of the m6A reader YTHDF2, but not YTHDF1 or YTHDF3, increases IAV replication and infectious particle production. We then used RNA:protein crosslinking and immunoprecipitation techniques to map the location of m6A residues on both the IAV mRNA/cRNA (plus) strands and vRNA (minus) strands and observed a high level of m6A addition on the viral mRNAs encoding the structural proteins HA, NA, M1/M2, and NP, but lower levels on the mRNAs encoding the viral polymerase proteins PB2, 378 Cell Host & Microbe 22, 377–386, September 13, 2017

PB1, and PA. Finally, we generated mutant forms of IAV in which eight prominent m6A sites on the HA mRNA/cRNA plus strand, or nine m6A sites on the HA vRNA minus strand, were silently mutated, and we observed that both these IAV mutants selectively expressed lower levels of HA mRNA and protein while expression of the other IAV mRNAs and proteins was unaffected. Moreover, these m6A-deficient IAV mutants were found to be significantly less pathogenic when introduced into mice. These data demonstrate that silent mutations that ablate m6A sites present on a viral RNA can significantly reduce viral gene expression in cis, therefore provide a potential explanation for the apparently ubiquitous presence of m6A residues on transcripts encoded by nuclear DNA and RNA viruses (Kennedy et al., 2017). RESULTS As noted above, it has previously been reported that IAV replication can be effectively inhibited using non-toxic does of the drug DAA, which has been reported to inhibit the addition of m6A residues to mRNA transcripts by inducing the depletion of SAM, the methyl donor for METTL3 (Bader et al., 1978; Fischer et al., 1990; Fustin et al., 2013). Although DAA is therefore not a highly specific inhibitor of m6A addition, this result, which is reproduced in Figure 1A, is nevertheless consistent with the hypothesis that m6A addition exerts a positive effect on IAV replication. To extend these data, we generated knock outs of the key

Figure 2. Overexpression of YTHDF2 Increases All Aspects of Viral Gene Expression (A) A549 cell lines overexpressing YTHDF1-Flag (Y1), YTHDF2-Flag (Y2.1 and Y2.2), or GFP were generated by lentiviral transduction followed by single cell cloning. (B) A549 cell lines were infected with IAV-PR8 at an MOI of 0.01 and expression of the viral proteins NP, NS1, and M2 assessed by western blot at 24, 48, and 72 hpi. GFP, YTHDF1, and YTHDF2 were detected using anti-Flag. The parental A549 cell line, and A549 cells expressing GFP-Flag, were used as controls. Quantification of the band intensities for NS1 and M2 at 72 hpi revealed expression levels of 6.28 ± 1.28 and 5.19 ± 2.13 for Y2.1 and 5.70 ± 1.65 and 5.84 ± 1.73 for Y2.2, respectively, when the expression in wild-type cells was normalized to 1.0. Actin was used as the loading control. Single round infections are described in Figure S2. (C) qRT-PCR was used to determine the mRNA levels of the spliced M2 IAV mRNA at the same time points post-infection. See Table S2 for primer sequences. (D) The viral titer produced from these cell lines at 72 hpi was determined by plaque assay on MDCK cells. These data represent the average of three biological replicates with SD indicated. *p < 0.05, **p < 0.01.

m6A writer METTL3 in the human lung epithelial cell line A549, using gene editing with CRISPR/Cas. We obtained two independent A549 cell clones that were defective for METTL3 expression based on both western blot (Figure 1B) and sequencing of all three copies of the METTL3 gene present in A549 cells (Figure S1). Analysis of the ability of these cells to support the replication of the IAV isolate PR8 revealed a substantial,
8-fold reduction in IAV gene expression measured by both western blot for the IAV NS1, NP, and M2 proteins (Figure 1C) and qRT-PCR analysis of the expression of the spliced IAV M2 RNA (Figure 1D). Similar data were obtained when expression of the IAV NP mRNA was quantified (data not shown). Moreover, we observed a highly significant (p < 0.01) reduction in the production of infectious IAV by the two METTL3 knockout cell lines M3.1 and M3.2, when compared with wild-type A549 cells or cells transduced with a lentiviral vector expressing GFP (Figure 1E). In conclusion, these data argue that m6A addition exerts a positive effect on IAV gene expression and virion production. Potent Activation of IAV Gene Expression by Ectopic YTHDF2 As noted above, m6A residues are bound by one of the three cytoplasmic reader proteins YTHDF1, YTHDF2, and YTHDF3

(Kennedy et al., 2017; Meyer and Jaffrey, 2014), and we have previously reported that overexpression of these proteins, in particular YTHDF2, significantly enhances HIV-1 gene expression and replication (Kennedy et al., 2016). To test whether this is also true for IAV, we generated clonal cell lines derived from A549 by transduction with lentiviral vectors expressing either YTHDF1 or YTHDF2 (Figure 2A). Following infection of these cell lines with IAV-PR8 at an MOI of 0.01, we observed an obvious increase in the level of expression of the IAV NS1, NP, and M2 proteins when assayed at 24, 48, and 72 hr post-infection (Figure 2B). We also detected a higher level of M2 mRNA (Figure 2C) and NP mRNA (data not shown) expression in the infected A549 cultures overexpressing YTHDF2, as measured by qRT-PCR, as well as a significant (p < 0.05) increase in the production of infectious progeny IAV virions (Figure 2D), when compared with wildtype A549 cells or A549 cells transduced with a control lentiviral vector expressing GFP. While both clonal A549 cell lines overexpressing YTHDF2 displayed this remarkable positive effect on IAV gene expression, we did not see any significant effect, either positive or negative, upon overexpression of YTHDF1 (Figure 2). As shown in Figure S2D, IAV also gave rise to significantly larger plaques when assayed on YTHDF2-overexpressing A549 cells, when compared with control cells or cells overexpressing YTHDF1, which argues that YTHDF2 overexpression not only increases IAV gene expression but also facilitates viral spread in culture. To confirm and extend these data looking at a spreading IAV infection (Figure 2), we also performed a single-cycle IAV Cell Host & Microbe 22, 377–386, September 13, 2017 379

replication assay using an MOI of 1.0 and in the absence of trypsin, which is required for IAV spread in culture. As shown in Figure S2A, we again detected a dramatic
16-fold increase in the expression of the IAV NS1 and M2 proteins in the A549 cells overexpressing YTHDF2, while YTHDF1 exerted at most a weak positive effect. In addition, we also performed single-cycle replication assays, again in the absence of trypsin, using an MOI of 3.0, which should result in the immediate infection of
93% of the cells, as was indeed confirmed by immunofluorescence (Figure S2C). This high MOI experiment (Figure S2B) again revealed a strong enhancement in the expression of the IAV NS1, M2, and NP proteins in the A549 cells overexpressing YTHDF2, when compared with control cells. These data (Figure S2A and 2B) therefore demonstrate that YTHDF2 can significantly increase the expression of IAV gene products in the absence of viral spread and thus argue that YTHDF2 is not acting solely by increasing the production of progeny IAV virions. Finally, as we had been unable to generate YTHDF3 overexpressing A549 cells using lentiviral transduction, we decided to generate YTHDF1, YTHDF2, and YTHDF3 overexpressing A549 cells using an entirely different technique, i.e., a dCas-based synergistic activation module targeted to the promoter of the endogenous YTHDF1, YTHDF2, or YTHDF3 genes (Konermann et al., 2015). The resultant clonal A549 cell lines only modestly overexpressed YTHDF1 or YTHDF2 relative to wild-type A549 cells, but we were able to derive two clonal cell lines overexpressing YTHDF3, which is normally almost undetectable in A549 cells (Figure S3). Analysis of the ability of these A549 cell clones to support IAV gene expression, by western blot for the IAV NS1 and M2 proteins, again revealed a strong induction by ectopic YTHDF2 but no obvious positive effect on IAV gene expression upon overexpression of YTHDF1 or, in this case, YTHDF3. Mapping m6A Sites on IAV mRNAs and vRNAs While the data presented so far clearly demonstrate that m6A addition and detection by YTHDF2 strongly enhances IAV replication, they do not demonstrate that this effect is direct. As noted above, IAV mRNAs have previously been reported to be highly m6A modified (Krug et al., 1976; Narayan et al., 1987), and we therefore decided to map these m6A sites using a previously described photoactivatable ribonucleosideenhanced crosslinking and immunoprecipitation (PAR-CLIP) approach (Hafner et al., 2010; Kennedy et al., 2016) to precisely define the binding sites of ectopically expressed YTHDF1, YTHDF2, or YTHDF3 in IAV-infected 293T cells that had been pulsed with 4-thiouridine (4SU), followed by RNase treatment of the immunoprecipitated RNA:protein complex, cDNA synthesis, and deep sequencing of the resultant YTHDF protein binding sites. As previously noted, a key advantage of the PAR-CLIP technique is that reverse transcription of the recovered short RNAs results in the insertion of characteristic T to C mutations at sites of protein crosslinking to 4SU residues, thus allowing the exclusion from the subsequent bioinformatics analysis of all background reads resulting from non-crosslinked RNAs (Kennedy et al., 2016; Hafner et al., 2010). As shown in Figure 3, we detected a large number of YTHDF protein binding sites on the viral mRNAs/cRNAs encoding the HA, NP, NA, and M open reading frames but 380 Cell Host & Microbe 22, 377–386, September 13, 2017

far fewer on the mRNAs/cRNAs encoding the PB2, PB1, and PA open reading frames. This apparent dichotomy is interesting as it suggests that m6A residues are selectively present on IAV mRNAs encoding the major viral structural proteins, which are expressed at high levels in IAV-infected cells, and less prevalent on the mRNAs encoding the three IAV RNAdependent RNA polymerase subunits, which are required at lower levels for optimal viral replication. Interestingly, this does not reflect the prevalence of potential 50 -RAC-30 m6A addition sites, which are evenly distributed across the IAV genome (data not shown). In addition to using PAR-CLIP with the three YTHDF proteins to map m6A residues in living, IAV-infected cells, we also used the distinct photo-crosslinking-assisted m6A sequencing (PA-m6A-seq) technique (Chen et al., 2015), which involves isolation of poly(A)+ mRNA from IAV-infected, 4SU-pulsed cells followed by binding of an m6A-specific antibody in vitro, crosslinking, RNase treatment, cDNA synthesis, and deep sequencing. These data, shown on the bottom row of Figures 3B and 3C, again confirmed that m6A residues were present on multiple locations of the viral mRNAs encoding HA, NP, M, and NA and at lower levels on the mRNAs encoding PB2, PB1, and PA. The mRNA encoding the IAV HA protein has previously been reported to contain eight internal m6A residues (Narayan et al., 1987) and we decided to focus on this unspliced RNA for our further research. As shown in Figure 3C, there is good concordance between the three PAR-CLIP experiments performed using the three YTHDF proteins and the data generated using the PA-m6A-seq technique in terms of the mapped location of m6A residues on the HA mRNA, and in fact our data identified eight major m6A peaks on the HA mRNA that were all located in the HA open reading frame (ORF), almost all of which could be inactivated by silent mutagenesis of the m6A consensus addition site 50 -RAC-30 (Figure S3). In addition to mapping m6A sites present on the IAV mRNA/ cRNA segments, we were also able to map m6A residues on the eight IAV vRNA minus strands using both PAR-CLIP with the YTHDF proteins and using PA-m6A-seq (Figure 4). The latter was unexpected, as PA-m6A-seq uses highly purified poly(A)+ RNA as the input RNA, and vRNAs are not polyadenylated. We hypothesize that viral vRNAs may have annealed to complementary poly(A)+ viral mRNAs during their isolation and, hence, been recovered along with the total cellular poly(A)+ RNA population. Strand origin was assigned based on the detection of A to G, versus T to C, mutations that arise from sites of protein crosslinking to incorporated 4SU residues. Regardless, the PAR-CLIP and PA-m6A-seq data obtained again revealed multiple m6A sites on the vRNA segments encoding the viral structural proteins HA, NP, NA, and M, and a lower level of m6A sites on the vRNA segments specific for PB2, PB1, and PA (Figure 4B). Importantly, these data again showed a strong concordance in the mapped locations of m6A residues on the HA vRNA segment and identified nine prominent m6A peaks (Figure 4C), all of which could be inactivated by mutagenesis of coincident consensus m6A addition sites (50 -RAC-30 ) in the HA vRNA segment. These nine introduced mutations were again designed to not affect the predicted coding capacity of the cognate HA mRNA (Figure S3).

Figure 3. Identification of m6A Sites on IAV-PR8 Plus-Sense mRNA PAR-CLIP and PA-m6A-seq were performed using 293T cell lines 24 hpi with IAV-PR8 at an MOI of 10. (A) Concatenated map of the IAV-PR8 transcriptome that reads were aligned to. (B) Complete transcriptome coverage tracks are shown for PAR-CLIP performed on Flag-GFP, Flag-YTHDF1-, Flag-YTHDF2-, and Flag-YTHDF3-expressing 293T cells, while PA-m6A-seq was performed using wild-type 293T cells. The PA-m6A-seq lane has a y axis of 0–500 reads, and all others are depicted with y axes of 0–200 reads. (C) An expanded view of the HA segment of IAV-PR8, with eight prominent m6A sites numbered. PA-m6A-seq has a y axis of 0–250 reads, and all others are depicted with y axes of 0–100 reads. Reverse transcription of crosslinked 4SU residues results in characteristic T > C mutations and the level of T > C conversion at specific residues is indicated by red/blue bars.

Loss of m6A Sites Present on the HA Segment of IAV Selectively Reduces HA Gene Expression Based on the m6A mapping data for the IAV HA RNA segment, we generated a full-length version of the HA gene segment lacking the plus-sense m6A sites mapped in Figure 3C, as well as a second HA gene segment lacking all nine minus-sense m6A sites mapped in Figure 4C (see Figure S3 for the precise point mutations introduced into HA). As noted above, these mutations, a total of 14 point mutations on the HA plus strand and 15 on the HA minus strand, all introduced synonymous codons into the encoded HA mRNA. Replication-competent IAV-PR8 mutants lacking m6A sites mapped on the HA plus-sense RNA (+Mut) or on the minus-sense RNA (–Mut) were then rescued by transfection of 293T cells and amplified in embryonated chicken eggs. We recovered comparable levels of the wild-type, +Mut, and –Mut IAV viruses from eggs, as confirmed by assay of

neuraminidase activity (Figure S4A), and these contained comparable levels of the HA vRNA segment (Figure 5A). However, analysis of purified IAV virions by western blot revealed a modest, 20%–40% drop in the incorporation of the HA protein into the –Mut and +Mut virions compared with wild-type IAVPR8 (Figure 5B). Quantification of HA mRNA expression using qRT-PCR after infection of A549 cells at an MOI of 0.01 also revealed a significantly lower level of HA mRNA expression (p < 0.01) for both the –Mut and +Mut IAV mutant viruses when compared with wild-type IAV-PR8 (Figure 5C). In contrast, the observed levels of other IAV mRNAs, typified here by the M2 mRNA, were not affected (Figure 5D). This demonstrates that the effect of these mutations on the expression of the HA mRNA is specific. An even more striking phenotype was observed when the A549 cells infected with wild-type and mutant IAV were analyzed for viral protein expression by western Cell Host & Microbe 22, 377–386, September 13, 2017 381

Figure 4. Identification of m6A Sites on IAV-PR8 Minus Strand vRNA PAR-CLIP and PA-m6A-seq were performed on RNA from 293T cell lines 24 hpi with IAV-PR8 at MOI of 10. (A) Concatenated map of the IAV-PR8 genome used for read alignment. (B) Complete genomic coverage tracks are shown, similar to Figure 2. The PA-m6A-seq lane has a y axis of 0–2000 reads, and all others are depicted with y axes of 0–300 reads. (C) An expanded view of the HA segment of IAV-PR8, with nine prominent m6A sites numbered. PA-m6A-seq has a y axis of 0–1,000 reads, and all others are depicted with y axes of 0–150 reads. Green/brown bars indicate the level of A > G conversion at specific A residues.

blot (Figure 5E). In this experiment, NS1 and M2 protein expression was similar for the wild-type and both mutant IAVs at 48 and 72 hr post-infection, and these two viral proteins therefore serve as internal controls. In sharp contrast, HA protein expression was down almost 2-fold for the +Mut virus and down
3-fold for the –Mut virus, and this difference was significant (p < 0.05). Therefore, we can conclude that the presence of m6A residues on both the IAV mRNA plus strands and vRNA minus strands has a direct, positive effect on IAV HA mRNA and protein expression. Our results also indicate that the observed reduction in HA protein expression does not inhibit the spread of IAV in embryonated chicken eggs (Figure S4A) and only modestly affects IAV spread in cultured A549 cells (Figure S4B). While the mechanism underlying the positive effect of m6A on HA expression is currently unknown, we considered the possibility that m6A residues on IAV transcripts might inhibit cellular 382 Cell Host & Microbe 22, 377–386, September 13, 2017

innate immune responses to viral infection, as has indeed been proposed (Durbin et al., 2016; Kariko et al., 2005). If this were the case, one would predict that infection with an IAV mutant lacking m6A sites would induce a higher level of expression of innate immune response genes such as RIG-I, MDA5 and/or interferon-b (IFN-b). Analysis of the expression of the mRNAs encoding these three immune effectors instead failed to reveal any positive effect of the m6A mutations introduced into the IAV HA segment (Figure 5F). Therefore, we conclude that the m6A sites present on the IAV HA mRNA and vRNA do not detectably downregulate the innate immune response to IAV infection. Loss of m6A Sites Present on the HA Segment of IAV Reduces Viral Pathogenicity In Vivo The modestly reduced level of HA mRNA and protein expression in the two m6A-deficient IAV mutants analyzed in culture in Figure 5 suggested that these mutants might also display

Figure 5. Mutagenesis of m6A Motifs on the IAV HA Segment Inhibits HA mRNA and Protein Expression Mutations introduced into the HA segment are described in Figure S3. (A) Viral RNA extracted from purified IAV-PR8 virions grown in embryonated chicken eggs was separated on a TBE-urea gel and stained. (B) Protein was extracted from purified IAV and western blotting used to determine HA protein levels in wild-type or mutant virions. Band intensity quantification revealed HA0 and HA1 levels of 0.8 ± 0.63 and 0.8 ± 0.54 for the +Mut virions and 0.5 ± 0.53 and 0.6 ± 0.48 for the –Mut virions, respectively, when HA levels in wild-type virions was normalized to 1.0. The viral protein M1 was used as a loading control. (C) A multicycle spreading infection was initiated by infection of A549 at an MOI of 0.01 using all three IAV-PR8 variants. Total RNA was extracted at 24, 48, and 72 hpi, and qRT-PCR then used to determine HA mRNA levels relative to a GAPDH mRNA internal control. See Table S2 for primer sequences. (D) The same total RNA samples used in (C) were used to quantify M2 mRNA levels, again using GAPDH mRNA as an internal control. (E) Similar to (C) except that western blotting was performed to evaluate the level of expression of the IAV HA, NS1, and M2 proteins at 24, 48, and 72 hpi. Quantification of HA band intensity at 72 hpi revealed expression levels of 0.69 ± 0.11 for the +Mut virus and 0.32 ± 0.13 for the –Mut virus, when normalized to M2 expression levels. Actin was used as the loading control. Viral spread was also determined by flow cytometry (see Figure S4). (F) A549 cells were infected at an MOI of 1.0 with the three IAV-PR8 variants and total RNA extracted 12 hpi. qRT-PCR was used to determine the levels of the cellular mRNAs encoding RIG-I, MDA5, and IFN-b1. All data are drawn from three biological replicates with SD indicated. ** = p < 0.01.

attenuated pathogenicity in vivo. To test this possibility, we infected mice with either 10 or 50 plaque-forming units (PFU) of either the parental IAV-PR8 isolate, which is highly pathogenic in mice, or of the –Mut and +Mut IAV-PR8 mutants, which have reduced m6A levels on the HA vRNA or mRNA strands, respectively. As may be observed (Figure 6), we saw reduced pathogenicity for both IAV mutants, but especially for the –Mut variant, at both the 50 (Figures 6C) and 10 PFU (Figure 6D) doses, with only the wild-type virus causing lethality at the lower dose, and this reduction was statistically significant at both infectious doses. In addition, we also saw reduced weight loss in the surviving mice infected with the two mutant viruses (Figures 6A and 6B). DISCUSSION Although work from several groups has demonstrated that a number of different DNA and RNA viruses express mRNAs that are modified by addition of m6A (Gonzales-van Horn and Sarnow, 2017; Kennedy et al., 2017), the role of m6A in regulating viral gene expression and replication remains largely unclear. We and others have reported that m6A addition to viral transcripts enhances the replication of HIV-1 (Kennedy et al., 2016; Lichinchi et al., 2016a), though a third group has disputed this finding (Tirumuru et al., 2016). In the case of the cytoplasmic

RNA virus HCV, m6A has been proposed to reduce the production of progeny virions without affecting viral RNA replication or protein expression (Gokhale et al., 2016) and, in the case of ZKV, enhanced virion production has also been observed upon knock down of m6A addition (Lichinchi et al., 2016b). However, given that m6A has been detected on transcripts expressed by a wide range of viral species (Kennedy et al., 2017) and, as sites of m6A addition are, at least in the case of HIV-1, evolutionarily conserved (Kennedy et al., 2016) we would argue that m6A must facilitate some aspect(s) of the viral life cycle. Especially for rapidly evolving viruses, such as HIV-1, IAV, or ZKV, one would expect the speedy selection of viral variants that had lost the consensus sites required for m6A addition if these indeed exerted an inhibitory effect in cis. In this manuscript, we have sought to address whether m6A affects the replication and pathogenicity of IAV, which was the first virus reported to bear multiple m6A residues (Krug et al., 1976). We initially addressed whether the global perturbation of m6A addition or recognition in the human lung epithelial cell line A549 would affect IAV replication, and we observed an
5-fold decrease in IAV protein expression and replication when the gene encoding the m6A methyltransferase METTL3 was inactivated by gene editing (Figure 1), and a similar R5-fold increase in IAV gene expression and virion production in A549 cells Cell Host & Microbe 22, 377–386, September 13, 2017 383

Figure 6. IAV Mutants Depleted for m6A on the Viral HA Strand Show Reduced Pathogenicity In Vivo Wild-type C57BL/6 mice were infected with 50 plaque-forming units (PFU) of wild-type (WT) PR8, +Mut, or –Mut and monitored for (A) body weight with 80% of the starting body weight as a humane endpoint, as indicated by the dashed line and (C) mortality. Similarly, mice were infected with 10 PFU of WT PR8, +Mut, or –Mut and monitored for (B) body weight and (D) mortality. Five mice were used per group. Significance is indicated on the graph based on the log rank Mantel-Cox test.

overexpressing the m6A reader protein YTHDF2 (Figure 2 and S2). In contrast, overexpression of YTHDF1 and YTHDF3 did not greatly affect IAV replication. To our knowledge, this is the largest increase in IAV replication ever observed upon simple overexpression of a single human protein in infected cells and argues that addition and detection of m6A plays a critical role in promoting IAV replication. Importantly, this marked positive effect was also observed under conditions where IAV spread was blocked (Figure S2A and B), thus arguing that YTHDF2 overexpression is not promoting IAV replication by, for example, enhancing the production of progeny virions but rather must be acting to promote viral gene expression and/or viral RNA replication. We note that the strong positive effect of YTHDF2 on IAV virion production (Figure 2D) may have practical importance given that difficulties with IAV vaccine production in embryonated chicken eggs have led to efforts to instead produce IAV vaccine strains in cultured mammalian cells, resulting in the recent licensing of the IAV vaccine Flucelvax (Houser and Subbarao, 2015). However, the production of IAV by cultured cells is relatively inefficient and the ability to substantially enhance this process by simple overexpression of YTHDF2 might therefore represent a significant technical advance. While the negative effect of METTL3 inactivation and the positive effect of YTHDF2 overexpression on IAV replication are both readily detectable (Figures 1 and 2), this could clearly result from the effect of m6A residues on the expression of cellular, rather than IAV, RNA transcripts. To address this concern, we therefore mapped m6A residues of both the plus-sense, mRNA/cRNA transcripts encoded by IAV (Figure 3) and on the minus sense, vRNAs (Figure 4). We then selected an unspliced, highly m6A-modified IAV gene segment, encoding the IAV HA protein, and generated mutant IAV-PR8 viral stocks in which m6A sites on the HA mRNA, or m6A sites on the IAV HA vRNA, were inactivated by mutation of 50 -RAC-30 m6A consensus sequences found coincident with the mapped sites of m6A addition (Figures 3 and 4). Importantly, although all 17 mapped m6A addition sites were located in the HA ORF, we were able to inactivate almost all of the 50 -RAC-30 consensus sequences by using synonymous mutations that do not affect the coding capacity of the HA 384 Cell Host & Microbe 22, 377–386, September 13, 2017

segment. It should be noted that these mutations do not remove all potential m6A addition sites found on the IAV HA segment (Figures 3 and 4), including two potential m6A sites on the viral mRNA strand that could not be silently mutated, so these mutant viruses are likely to be m6A hypomethylated on the HA segment, rather than totally devoid of m6A. Initial analysis showed that while these two viral mutants replicated in embryonated chicken eggs to similar levels as the wildtype IAV-PR8 virus (Figures 5A and S4A), the resultant IAV virions bore slightly lower levels of the HA protein (Figure 5B). When used to infect wild-type A549 cells in culture, the mutant IAVs lacking m6A on HA mRNAs (+Mut) or vRNA (–Mut) both expressed lower levels of the HA mRNA (Figure 5C), while the expression levels of other IAV mRNAs was unaffected (Figure 5D). Consistent with these RNA data, the IAV mutants lacking m6A residues on the HA transcripts also expressed lower levels of the viral HA protein relative to the IAV M2 and NS1 proteins expressed in the same infected cells, which again were unaffected (Figure 5E). These data are therefore consistent with the hypothesis that m6A editing of the HA mRNA and vRNA transcripts directly enhances HA RNA replication and/or expression, which is consistent with the finding that YTHDF2 overexpression also enhances IAV gene expression under conditions where viral spread cannot occur (Figure S2). Importantly, when we analyzed the pathogenic potential of these IAV-PR8 mutants, which bear reduced levels of m6A on the HA strand but are otherwise wildtype, we observed a statistically significant reduction in the pathogenic potential of both the +Mut and –Mut viruses (Figure 6). This suggests that an IAV variant lacking m6A residues on all eight gene segments would likely be highly attenuated and also suggests that the addition of m6A might be a good target for the potential development of a broad spectrum antiviral that inhibits not only IAV replication but also the replication of some of the other viruses reported to bear m6A residues on their transcripts. Indeed, the drug DAA, which has been reported to inhibit m6A addition by depleting intracellular SAM levels is a potent inhibitor of not just IAV replication (Figure 1A), but also a range of other viruses at doses that are non-toxic in animals (Bray et al., 2000; Wyde et al., 1990). Because m6A and other epitranscriptomic changes have been proposed to inhibit the detection of foreign RNA molecules by cellular innate immune effectors (Durbin et al., 2016; Kariko et al., 2005), we addressed whether the reduced addition of

m6A residues to HA transcripts might result in the enhanced activation of RIG-I, MDAS, or IFN-b mRNA expression; however, this was not observed. Similarly, inactivation of METTL3 or overexpression of YTHDF2 did not exert an obvious effect on the basal level of expression of innate immune effectors in A549 cells (data not shown). It remains possible that elimination of all m6A residues on the IAV genome might give a different result. The data presented in this manuscript do not define the mechanism by which m6A enhances IAV gene expression and replication, but do eliminate some possibilities. Because m6A addition enhances the expression of both spliced (NS1 and M2) and unspliced (NP) IAV gene segments equivalently, it is unlikely that m6A acts by regulating splicing. Also, because the increase in IAV protein expression closely matches the increase in mRNA expression (Figures 1 and 2), it is unlikely that the positive effect of m6A on IAV gene expression is due to enhanced translation. Finally, as m6A residues present in cis enhance the function of not only the HA mRNA but also the HA vRNA, which is confined to the cell nucleus until packaged into progeny virion particles late in the viral life cycle, an effect of m6A on nuclear RNA export also seems unlikely. Instead, m6A seems to be acting by increasing IAV RNA levels by either enhancing viral replication or by enhancing viral RNA stability. Interestingly, while m6A has been reported to destabilize RNAs in some settings (Ke et al., 2017; Wang et al., 2014), others have reported that m6A can act to stabilize mRNAs (Fry et al., 2017). Of note, we have previously reported that m6A residues enhance the expression level of HIV-1 mRNAs as well as indicator plasmid mRNAs bearing m6A residues in cis, which may suggest that m6A is used by both IAV and HIV-1 to increase the steady-state level of viral mRNA expression by a similar, post-transcriptional mechanism. Certainly, it seems very unlikely that these viruses would have retained m6A residues on their transcripts if these indeed exerted a destabilizing effect. In conclusion, our data argue that m6A residues act in cis to promote the expression of IAV transcripts and that inhibition of m6A addition to IAV RNAs either indirectly, by elimination of METTL3, or directly, by elimination of sites of m6A addition on the IAV HA transcript, therefore results in a drop in not only viral gene expression and replication but also pathogenicity. Precisely how m6A exerts this strong positive effect on the replication of the nuclear RNA virus IAV, and how the divergent data obtained with the cytoplasmic RNA viruses HCV and ZKV, suggesting inhibition of viral spread by m6A (Gokhale et al., 2016; Lichinchi et al., 2016b), can be reconciled remains to be determined. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell Lines B YTHDF Overexpressing A549 Cell Lines B METTL3 Knock-Out A549 Cell Lines B CRISPR SAM Expressing Cell Lines B Animal Subjects

d

d d

METHOD DETAILS B Western Blots B IAV Infections B Quantitative RT-PCR B IAV Plaque Assays B PAR-CLIP and PA-m6A-Seq Analysis B Small RNA Sample Preparation and Deep Sequencing B Bioinformatics B Rescue of IAV PR8 Mutants B Immunofluorescent Cell Staining B A549 Multicycle Infection Flow Cytometry B Animal Experiments QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.chom.2017.08.004. AUTHOR CONTRIBUTIONS D.G.C., E.M.K., H.P.B., R.E.D., N.S.H., and B.R.C. designed experiments, analyzed the data, and wrote the manuscript. D.G.C., E.M.K., K.T., H.P.B., and R.E.D. performed the experiments. ACKNOWLEDGMENTS This research was supported by NIH grant R21-AI130574. D.G.C. was funded by Marie-Sk1odowska Curie Global Fellowship MSCA-IF-GF:747810. K.T. was supported by NIH grant T32-CA009111 and R.E.D. was supported by T32GM007184. The authors thank Feng Zhang for reagents used in this work. Received: April 27, 2017 Revised: July 3, 2017 Accepted: August 8, 2017 Published: September 13, 2017 REFERENCES Bader, J.P., Brown, N.R., Chiang, P.K., and Cantoni, G.L. (1978). 3-Deazaadenosine, an inhibitor of adenosylhomocysteine hydrolase, inhibits reproduction of Rous sarcoma virus and transformation of chick embryo cells. Virology 89, 494–505. Batista, P.J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D.M., Lujan, E., Haddad, B., Daneshvar, K., et al. (2014). m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15, 707–719. Boden, D., Pusch, O., Lee, F., Tucker, L., and Ramratnam, B. (2003). Human immunodeficiency virus type 1 escape from RNA interference. J. Virol. 77, 11531–11535. Bray, M., Driscoll, J., and Huggins, J.W. (2000). Treatment of lethal Ebola virus infection in mice with a single dose of an S-adenosyl-L-homocysteine hydrolase inhibitor. Antivir. Res 45, 135–147. Chen, K., Lu, Z., Wang, X., Fu, Y., Luo, G.Z., Liu, N., Han, D., Dominissini, D., Dai, Q., Pan, T., et al. (2015). High-resolution N(6) -methyladenosine (m(6) A) map using photo-crosslinking-assisted m(6) A sequencing. Angew. Chem. Int. Ed. 54, 1587–1590. Das, A.T., Brummelkamp, T.R., Westerhout, E.M., Vink, M., Madiredjo, M., Bernards, R., and Berkhout, B. (2004). Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J. Virol. 78, 2601–2605. Desrosiers, R.C., Friderici, K.H., and Rottman, F.M. (1975). Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5’ terminus. Biochemistry 14, 4367–4374.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Anti-Flag (1:5000)

Sigma

Cat# F1804 RRID: AB_262044

Anti-Haemaglutinin (1:5000)

Mount Sinai

PY102

Anti-NS1(1:1000)

Santa Cruz

Cat# sc-130568 RRID: AB_2011757

Antibodies

Anti-Matrix (1:5000)

Kerafast

Cat# E10 RRID: AB_2687434

Anti-NP (1:1000)

AbD

Cat# MCA400 RRID: AB_2151884

Anti-Actin (1:10000)

Santa Cruz

Cat# sc-47778 RRID: AB_626632

Anti-YTHDF1 (1:2000)

Abcam

Cat# ab99080 RRID: AB_10675362

Anti-YTHDF2 (1:2000)

Proteintech

Cat# 24744-1-AP RRID: AB_2687435

Anti-YTHDF3 (1:2000)

Santa Cruz

Cat# sc-377119 RRID: AB_2687436

Anti-Mettl3 (1:2000)

Abnova

Cat# H00056339-B01P RRID: AB_2687437

Anti-Mouse HRP (1:10000)

Sigma

Cat# A9044 RRID: AB_258431

Anti-Rabbit HRP (1:10000)

Sigma

Cat# A6154 RRID: AB_258284

Alexa Fluor 488 Anti-mouse

Thermo

Cat# A-11029 RRID: AB_138404

Anti-m6A

Synaptic Systems

Cat# 202 111 RRID: AB_2619891

Anti-PR8

In house

N/A

Influenza A - PR8

This Study

N/A

Influenza A - PR8 plus strand mutant

This study

N/A

Influenza A - PR8 negative strand mutant

This study

N/A

DH5a

NEB

Cat# C2988J

Bacterial and Virus Strains

Biological Samples Blasticidin S

Gemini Bio-Products

Cat# 400-165P

Hygromicin B

Corning

Cat# 30-240-CR

PEI

Polysciences

Cat# 23966-2

Puromycin

Gemini Bio-Products

Cat# 400-128P

3-deazaadenosine (DAA)

Sigma

Cat# D8296

4-thiouridine (4SU)

Carbosynth

Cat# NT06186

BSA

Sigma

Cat# A9576

NaHCO3

Thermo

Cat# 25080094

TPCK-treated Trypsin

Sigma

Cat# T1426

16% Paraformaldehyde

Thermo

Cat# 28908

Oxoid Agar

Thermo

Cat# LP0011B

True Blue HRP Substrate

VWR

Cat# 95059-468

Laemmli Buffer

Cold Spring Harbor

http://cshprotocols.cshlp.org/content/2006/1/ pdb.rec10424

WesternBright ECL kit

Advansta

Cat# K-12045-D50

Live/Dead fixable violet stain

Thermo

Cat# L34955

Critical Commercial Assays TruSeq Small RNA Sample Preparation Kit

Illumina

Cat# 15016911

SuperScript III

Invitrogen

Cat# 18080-044

GoTaq green PCR master mix

Promega

Cat# M7123

Power Sybr Green PCR Mastermix

Applied Biosystems

Cat# 4367659 (Continued on next page)

Cell Host & Microbe 22, 377–386.e1–e5, September 13, 2017 e1

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

This study

https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE98033

Deposited Data PAR-CLIP and PA-m6A-seq deep Experimental Models: Cell Lines HEK293T

ATCC

ATCC Cat# CRL-11268, RRID:CVCL_1926

A549

ATCC

ATCC Cat# CRM-CCL-185, RRID:CVCL_0023

MDCK

ATCC

ATCC Cat# CCL-34, RRID:CVCL_0422

HEK293T-pLEX:YTHDF1

Kennedy et al., 2016

N/A

HEK293T-pLEX:YTHDF2

Kennedy et al., 2016

N/A

HEK293T-pLEX:YTHDF3

Kennedy et al., 2016

N/A

A549-pLEX:YTHDF1

This study

N/A

A549-pLEX:YTHDF2.1

This study

N/A

A549-pLEX:YTHDF2.2

This study

N/A

A549-CRISPR:Mettl3.1

This study

N/A

A549-CRISPR:Mettl3.2

This study

N/A

A549-SAM:YTHDF1.1

This study

N/A

A549-SAM:YTHDF1.2

This study

N/A

A549-SAM:YTHDF2.1

This study

N/A

A549-SAM:YTHDF2.2

This study

N/A

A549-SAM:YTHDF3.1

This study

N/A

A549-SAM:YTHDF3.2

This study

N/A

Jackson Laboratories

WT C57BL/6

For sgRNA sequences see Table S1

This study

N/A

For qRT-PCR primer sequences see Table S2

This study

N/A

MS2-p65-HSF1

Addgene

Cat# 61426

DCR8.74

Addgene

Cat# 22036

pMD2.G

Addgene

Cat# 12259

lentiSAMv2

Addgene

Cat# 75112

lentiCRISPR v2

Addgene

Cat# 52961

pLex-YTHDF1

Kennedy et al., 2016

N/A N/A

Experimental Models: Organisms/Strains Female WT C57BL/6 mice Oligonucleotides

Recombinant DNA

pLEX-YTHDF2

Kennedy et al., 2016

HA gBlocks

IDT

N/A

pDZ

Quinlivan et al., 2005

N/A

pDZ-PR8:HA positive mutant

This study

N/A

pDZ-PR8:HA negative mutant

This study

N/A

Software and Algorithms Bowtie

Langmead et al., 2009

http://bowtie-bio.sourceforge.net/index.shtml

Fastx Toolkit

Cold Spring Harbor

http://hannonlab.cshl.edu/fastx_toolkit/ index.html

Samtools

Li et al., 2009

http://samtools.sourceforge.net

Other Tris-Glycine SDS PAGE gel

Invitrogen

Cat# WT4202BOX

10% (wt/vol) polyacrylamide gels

Biorad

Cat# 3450052

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CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to, and will be fulfilled by, the Lead Contact, Bryan R. Cullen ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Lines The human 293T and A549 cell lines, and canine MDCK cells, were obtained from the ATCC. Wildtype and modified versions of these cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics at 37 C in 5% CO2. The 293T cell lines expressing ectopic, FLAG-tagged YTHDF proteins were previously published (Kennedy et al., 2016). The 293T and MDCK cell lines are of female origin, while the A549 cell line is of male origin. YTHDF Overexpressing A549 Cell Lines Lentiviral vectors, based on pLEX, expressing FLAG-tagged cDNAs encoding human YTHDF1 and YTHDF2, or GFP (Kennedy et al., 2016), were used to generate A549 cells that overexpress these proteins. 293T cells (2.5 x 106) were transfected using the PEI method with a lentiviral vector (15 mg), the packaging plasmid DCR8.74 (10 mg), and the VSV-G envelope expression plasmid pMD2.G (5 mg) to generate lentiviral particles that were used to transduce A549 cells. After puromycin (2 mg/ml) selection, the transduced cells were single cell cloned and cell lines expressing high levels of FLAG-tagged YTHDF1, YTHDF2 or gfp identified by Western blot. One clonal YTHDF1 and two clonal YTHDF2 overexpressing A549 cell lines were generated, termed A549/Y1, A549/Y2.1 and A549/Y2.2. METTL3 Knock-Out A549 Cell Lines CRISPR/Cas9 was used to knock-out the METTL3 gene in A549 cells. An sgRNA specific for exon 4 of the METTL3 gene (Table S1) was obtained from the GECKO sgRNA library (Sanjana et al., 2014) and cloned into the lentiviral vector lentiCRISPRv2. 293T cells (2.5 x 106) were transfected using the PEI method with the lentiviral vector expressing both the sgRNA and Cas9 (15 mg), the packaging plasmid DCR8.74 (10 mg), and the VSV-G envelope expression plasmid pMD2.G (5 mg) to generate lentiviral particles that were used to transduce A549 cells. After puromycin (2 mg/ml) selection, the transduced cells were single cell cloned and cells lacking METTL3 expression identified by Western blot. Genome editing of the targeted region was also confirmed by PCR of exon 4 of METTL3 from genomic DNA followed by sequencing (Figure S1). Two METTL3 knockout A549 cell lines were generated, termed A549/M3.1 and A549/M3.2. CRISPR SAM Expressing Cell Lines 293T cells (2.5 x 106) were transfected using the PEI method with a lentiviral vector expressing MS2-p65-HSF1 (15 mg) (Konermann et al., 2015), the packaging plasmid DCR8.74 (10 mg), and the VSV-G envelope expression plasmid pMD2.G (5 mg) to generate lentiviral particles used to transduce A549 cells. After hygromicin B (25 mg/ml) selection, the transduced cells were single cell cloned and a cell line expressing a high level of MS2-p65-HSF1 identified. This A549/MS2-p65-HSF1 cell line was then transduced with lentiviral particles generated in wildtype 293T cells transfected with lentiSAMv2, DCR8.74, and pMD2.G, as described above. LentiSAMv2 expresses both a dCAS9/VP64 fusion protein and an sgRNA expressed from a U6 promoter. Transduced cells were then selected for both blasticidin (10 mg/mL) and hygromycin B (25 mg/mL) resistance to generate polyclonal cell lines expressing dCas9/VP64, and MS2-p65-HSF1 and an sgRNA specific for the endogenous human promoter driving expression of YTHDF1, YTHDF2 or YTHDF3 (Table S1). A cell line transduced with a lentiviral vector lacking any sgRNA was used as a no guide control. A549-based cell lines overexpressing each YTHDF protein were then obtained by single cell cloning and overexpression of the YTHDF proteins verified by Western blot. The sgRNA sequences used were obtained from Table S6 from Konermann et al., 2015. Animal Subjects Female wildtype C57BL/6J mice were purchased (000664, Jackson Labs) and infected at 6 weeks old. All mice were maintained on a 12 h light/ dark cycle with continuous access to food and water. Experiments were performed at biosafety level 2 at Duke University. All procedures involving animals were performed as approved by the Duke University IACUC. METHOD DETAILS Western Blots All Western blots were performed using the same protocol. Protein samples were extracted using Laemmli buffer, sonicated and denatured at 95 C for 10 min before being loaded onto a Tris-Glycine SDS polyacrylamide gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane, and then blocked in 5% milk in PBS-Tween. Membranes were incubated in primary and secondary antibodies diluted in 5% milk/PBS-Tween, with membranes washed 3 times for 15 min each with PBS-Tween. Dilutions for each antibody are listed in the Key Resources Table. Chemiluminescence was visualized using WesternBright ECL.

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IAV Infections Both 293T and A549 cells were infected with wild type and mutant IAV-PR8 in Optimem supplemented with 1% BSA. Single round infections were performed at an MOI of 1.0 or MOI of 3.0 where indicated, while multicycle, spreading infections were performed at an MOI of 0.01. Cells were incubated with infectious media at 37 C for 2 h before this was replaced with DMEM supplemented with 1% bovine serum albumin (BSA) and antibiotics. For multicycle infections, post-infection media were also supplemented with TPCKtreated Trypsin. Quantitative RT-PCR qRT-PCR was performed to determine the mRNA levels for IAV genes M2 and HA, and cellular immune response genes RIG-I, MDA5 and IFNb1. The level of GAPDH mRNA was used to normalize all qRT-PCR experiments. All primer sequences are listed in Table S2. For multicycle IAV infections, RNA was collected at 0, 24, 48 and 72 hpi. For the evaluation of immune response genes, RNA was collected at 12 hpi. RNA was extracted using the TRIzol method. cDNA was generated using the Ambion cDNA synthesis kit following the manufacturer’s protocol. All cDNAs were generate with random primers, except for detection of IAV HA mRNA, which utilized an HA specific RT primer (HA.RT; Table S2). This primer was designed to reverse transcribe only HA mRNA, using the methodology published by Kawakami et al. (2011), by adding a non-viral tag CCAGATCGTTCGAGTCGT to the mRNA sequence, which was then used to specifically amplify HA mRNA. All qPCR was performed using Sybr Select Master Mix following the manufacturer’s instructions. All qRT-PCR was quantified using the DDCT method. IAV Plaque Assays Wild type and mutant IAV-PR8 was grown in embryonated chicken eggs using standard protocols, and the titer determined by plaque assay on MDCK cells. MDCK cells were grown in 6-well plates to 80-90% confluency. Serial dilutions of the IAV-PR8 stock were then overlaid on the MDCK cells and incubated at 37 C for 1h. Infectious media were then removed and replaced with plaque media, consisting of MEM, 5% NaHCO3, 1% BSA, 1mg/ml TPCK-treated Trypsin and 1% agar. MDCK cells were then incubated at 37 C for 72 h. Cells were fixed with 4% Paraformaldehyde (PFA) for 1h and the agar was washed off the wells. Cells were incubated with a polyclonal anti-PR8 antibody in 5% milk and PBS-Tween for 1 h, then washed 3 times for 5 min each with PBS-Tween and incubated with anti-mouse HRP in 5% milk and PBS-Tween for 1 h. Cells were then washed 3 times for 5 min each with PBS-Tween before being incubated with True Blue HRP Substrate for 15 min. The viral titer was ascertained by multiplying the number of plaques by the dilution factor. Plaque diameter was determined using ImageJ. PAR-CLIP and PA-m6A-Seq Analysis PAR-CLIP was performed as previously described (Hafner et al., 2010). 293T-based cell lines expressing FLAG-tagged YTHDF proteins, or FLAG-tagged GFP as a control (Kennedy et al., 2016), were pulsed with 100 mM 4SU in fresh media for 4 h. Cells were then infected with IAV-PR8 in 4SU-supplemented media for 4 h before media were replaced with 4SU-supplemented fresh media and incubated for a further 20 h. The cells were then UV irradiated, harvested and the PAR-CLIP protocol performed using an antibody specific for the FLAG epitope tag. For PA-m6A-seq, 293T cells were again pulsed with 4SU as described above. At the end of the 4SU pulse, total RNA was extracted using TRIzol, and poly(A)+ purified using oligo-dT. 10 mg of poly(A)+ RNA was then used for the standard PA-m6A-seq protocol (Chen et al., 2015) using an m6A specific polyclonal antibody. Small RNA Sample Preparation and Deep Sequencing RNA isolated from either PAR-CLIP or PA-m6A-seq was processed with the TruSeq Small RNA Sample Preparation Kit. Adapterligated RNA was reverse-transcribed using SuperScript III and then amplified with GoTaq green PCR master mix with the TruSeq 30 indices. PCR products were separated by gel electrophoresis on 10% (wt/vol) polyacrylamide gels and DNA bands corresponding to the expected
145 bp libraries isolated. PAR-CLIP and PA-m6A-seq libraries were sequenced on a HiSeq 2000. Base calling was performed with CASAVA and was processed with the fastx toolkit. Reads with a length greater than 14 bp were used for downstream bioinformatic analysis. Bioinformatics Read alignments were performed using Bowtie (Langmead et al., 2009). Reads were first aligned to the human genome build hg19 allowing up to 1 mismatch, then unaligned reads were aligned to the IAV-PR8 transcriptome, again allowing up to 1 mismatch. Characteristic T>C mutations, resulting from 4SU incorporation and crosslinking, were present among the viral aligned reads. In addition, A>G mutations were also present, corresponding to negative strand alignments. Reads containing T>C mutations were grouped as positive strand mRNA reads, while those containing A>G mutations were grouped as negative strand vRNA reads. All data was processed using in-house Perl scripts and Samtools (Li et al., 2009), and visualized with IGV, as previously described (Kennedy et al., 2016).

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Rescue of IAV PR8 Mutants A/Puerto Rico/8/1934 was used with a sequence corresponding to those deposited under accession numbers AF389115-389122 with the following changes. Point mutation T>C.1082 in segment 5. Segments were cloned into the ambisense pDZ vector by Gibson cloning, as previously described (Quinlivan et al., 2005). Mutant PR8 viruses lacking m6A sites were generated using the same sequences. Eight, on the plus strand, or nine, on the minus strand, silent mutations were introduced into the IAV-PR8 HA segment to remove DRACH motifs shown to be methylated by PARCLIP and PA-m6A-seq (Figure S3). Two gene blocks containing the full length IAV-PR8 HA segment containing these mutations, either on the plus or minus strand, were cloned into the ambisense pDZ vector by Gibson cloning, as previously described (Quinlivan et al., 2005). These IAV-PR8 mutants were then rescued using previously described methods (Heaton et al., 2016). Briefly, pDZ clones for each IAV segment and either the wild type, plus strand mutant or minus strand mutant pDZ-HA clone were transfected into 293T cells. Virus was amplified in embryonated chicken eggs. All wild type and mutant IAV-PR8 variants were plaque-purified, and HA segments sequenced to confirm the introduction of mutations, prior to phenotypic analysis. Mutant PR8 variants were titered using a Neuraminidase assay (Sigma), following the manufacturer’s instructions. To visualize packaged vRNA segments, IAV virions were isolated from embryonated eggs, overlaid on a 30% sucrose gradient and centrifuged at 27,500rpm for 90 min. Viral RNA was extracted using TRIzol and the vRNAs resolved on a 4% TBE Urea gel and then stained with ethidium bromide. Immunofluorescent Cell Staining A549 cell lines were grown on coverslips and infected with IAV-PR8 at an MOI of 3 for a single round infection, as described above. After 24 hpi media was removed and cells were fixed in 4% PFA at room temperature for 20 min. PFA was removed and the cells were washed in PBS. Cells were permeabilized in 0.1% Triton-X before being blocked in 1% BSA/PBS for 30 min under gentle agitation at room temperature. Cells were then probed with anti-NP at a 1:100 dilution in 1% BSA/PBS for 1 h under gentle agitation. Cells were washed for 10 min 3 times with PBS before being probed with anti-mouse Alexa Fluor 488 at a dilution of 1:1000 in 3% BSA/PBS for 1 h under gentle agitation. Then cells were again washed for 10 min 3 times in PBS before the coverslips were mounted on slides using Vectashield mounting media and imaged. A549 Multicycle Infection Flow Cytometry A549 lung epithelial cells were infected at an MOI of 0.01 and then incubated at 37 C in serum-free media containing 0.3 mg/mL trypsin to facilitate a multicycle infection. Samples were collected at the indicated timepoints, stained for live cells (ThermoFisher Live/Dead fixable violet stain), then fixed in 2% paraformaldehyde for 10 min at room temperature. After all samples were collected, they were stained using PY102 (provided by Tom Moran at the Experimental Therapeutics Institute at the Icahn School of Medicine at Mount Sinai) at 1 mg/mL in PBS/BSA. Samples were analyzed on a Fortessa X20 (BectonDickinson) then processed using FlowJo software. Animal Experiments At 6 weeks old, female wild type C57BL/6J mice were infected intranasally at the indicated doses of the wildtype IAV-PR8 isolate, +Mut or -Mut viruses in 40 mL of pharmaceutical grade PBS under anesthesia (ketamine/xylazine). Mouse morbidity was monitored via daily weighing, and a loss of >20% of the starting weight was defined as the humane endpoint. For all experimental conditions, 5 mice were used per dose. QUANTIFICATION AND STATISTICAL ANALYSIS Band intensities for Western blots were quantified using ImageJ. For qRT-PCR assays and titering of IAV-PR8, a Student’s t-test was performed to determine significance with p<0.05 deemed as statistically significant. For all qRT-PCR experiments 3 independent biological samples were collected and analyzed, with 3 experimental replicates for each sample. For Western blotting 3 independent biological samples were collected and analyzed. For immunofluorescent cell staining 3 independent biological replicates were stained and imaged. For flow cytometry 3 independent biological samples replicates were collected and analyzed, with 2 experimental replicates for each sample. DATA AND SOFTWARE AVAILABILITY The deep sequencing datasets generated in this study have been deposited in the GEO database under accession number GSE98033.

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