1. Volume 22, Issue 3, Pages 377-386.e5 (September 2017)
Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication 
David G. Courtney, Edward M. Kennedy, Rebekah E. Dumm, Hal P. Bogerd, Kevin Tsai, Nicholas S. Heaton, Bryan R. Cullen 
Cell Host & Microbe 
Volume 22, Issue 3, Pages e5 (September 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 22, 377-386. e5DOI: (10. 1016/j. chom. 2017
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3. 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.
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4. 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.
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5. 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.
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6. 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.
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7. 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-β1.
All data are drawn from three biological replicates with SD indicated. ∗∗ = p < 0.01.
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8. 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.
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