Fig 1.
(A) HEK293T cells were infected with PR8 virus at a multiplicity of infection (MOI) of 0.01 or transfected with Flag-NS1 and vector for 24 hours, cell lysates were immunoprecipitated with an anti-Flag or anti-NS1 antibody and then analyzed by silver stain. (B-C) HEK293T cells were transfected with the indicated plasmids for 24 h. The cell lysates were treated with or without 100U RNase A/T at 37°C for 30 min. CoIP assay was performed using an anti-HA (B) and anti-Flag (C) antibody and analyzed by western blotting. (D) A549 cells were infected with the PR8 virus at an MOI of 0.01 for 24 hours. The cell lysates were treated with or without 100U RNase A/T at 37°C for 30 min. CoIP assay was performed using an anti-YTHDC1 antibody. Input and IP complexes (NS1 and YTHDC1) were analyzed by western blotting. (E-G) A549 cells were infected with the influenza virus at MOI of 5. Cells were fixed and analyzed for the colocalization of YTHDC1 with NS1 after 3 and 6 hours post infection. Scale bar, 20 μm. (F and G) The normalized fluorescence of YTHDC1 and NS1 along the white arrowheads shows overlapping peaks.
Fig 2.
YTHDC1 promotes PR8 virus replication.
(A) METTL3, YTHDF2, and YTHDC1 were knocked down in A549 cells by shRNA, and then infected with the PR8 virus. Virus titer and protein expression were detected by TCID50 and western blotting at 24 hpi (left). Each protein band was quantified by ImageJ and normalized to GAPDH levels (right). Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; **, P<0.01). (B) WT and YTHDC1 knockdown A549 cells were transfected with 0.5 μg YTHDC1 for 24 hours, vector as a control, and then infected with PR8 virus (MOI = 0.01) for 24 h. Virus titer was detected by TCID50 assay (left). Each protein band was quantified by ImageJ and normalized to GAPDH levels (right). Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (Student t-test; ns, not significant; ***, P<0.001). (C) A549 cells were infected with the PR8 virus at an MOI of 0.01. Cell culture supernatants were collected at 12, 24, and 36 hpi. Virus titers were determined by TCID50 assay on MDCK cells. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (two-way ANOVA test; * P<0.05, **, P<0.01). (D) YTHDC1 knockdown (shYTHDC1) or negative control (shNC) knockdown A549 cells were infected with the PR8 virus at an MOI of 5. Cell lysates were subjected to western blotting analysis at 3, 6, and 9 hpi. GAPDH was used as a loading control. Each protein band was quantified by ImageJ and normalized to GAPDH levels. The YTHDC1/GADPH protein ratio was calculated. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; ns, not significant; *, P<0.05; ***, P<0.001).
Fig 3.
NS1 inhibits NS segment splicing by YTHDC1.
(A) YTHDC1 knockdown (shYTHDC1) or negative control (shNC) knockdown A549 cells were infected with the PR8 virus at an MOI of 5. The ratios of NEP to NS1 mRNA were calculated at indicated time points with the specific primers. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (two-way ANOVA test; * P<0.05, ** P<0.01; ***, P<0.001). (B) HEK293T cells were transfected with indicated plasmids for 24 h and then infected with the PR8 virus at an MOI of 1 for 12 h. RIPs and RT-qPCR were performed using specific primers detecting viral NS mRNA. Fold enrichment of mRNA was calculated. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; ***, P<0.001). (C) A549 cells were infected with the PR8 virus at an MOI of 1 for 12 h. RIPs and RT-qPCR were performed using specific primers detecting viral NS mRNA. Fold enrichment of mRNA was calculated. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; ***, P<0.001). (D) NS1 mRNA and its alternatively spliced product were depicted, and the arrowheads show primer positions for detection of various. G/GAC (528–531) is the YTHDC1-binding site and ‘/’ is the 3′ splicing site in the NS segment. (E) A view of the NS segment of IAV-PR8. PA-m6A-seq has a y-axis of 0–250 reads[14]. The 530A site is marked in the red box. (F) Analysis of GGAC motif in NS 528–531 segment of IAVs, total of 28189 strains were analyzed. (G) Pulldown YTHDC1 proteins using 20 bp NS biotin-probes of PR8: probes were immunoprecipitated with the cell lysis. And the bead eluate was analyzed by western blotting.
Fig 4.
NS1 regulates NS segment splicing through YTHDC1.
(A) Schematic illustration of NS-null minigenome system and detection of specific NS mRNAs. Construction of the pHI NS-null plasmid, which contains multiple stop codons (red triangle). Primers used for NS-null mRNA detection are shown as blue arrowheads. 3P (PA, PB1, PB2). NS-null (two stop codons were mutated at positions 45 and 456, with transcription but not translation). (B) HEK293T cells were transfected with the indicated viral RNP reconstitution plasmids (pCDNA-3.1-PB1, -PB2, -PA, and -NP, pPolI-Luc, and Renilla); polymerase activity was measured at 24 h post transfection. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (Student t-test; ***, P<0.001). (C) HEK293T cells were transfected with the minigenome system and Flag-NS1 or Flag-NS138/41A for 24 h, total RNAs were extracted and quantified by RT-qPCR with specific primers, and NEP/NS1 mRNA was calculated. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; ***, P<0.001). (D) HEK293T cells were transfected with the indicated plasmids for 24 h. CoIP assay was performed using an anti-Flag antibody and analyzed by western blotting. (E) YTHDC1 was knocked down by shRNA on HEK293T cells. And indicated plasmids were transfected with the minigenome system, and related NEP/NS1 ratios were detected at 24 hours post transfection. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; *, P<0.05; **, P<0.01).
Fig 5.
NSA530C mutation altered virus splicing and replication.
(A)A549 cells were infected with the wild type or mutated PR8 virus at an MOI of 1 for 12 h, and the mRNAs were purified with either the control IgG or the YTHDC1 antibody. The purified RNAs were then analyzed by specific RT-qPCR assay. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; ns, not significant; ***, P<0.001). (B) A549 cells were infected with either the wild type or mutant of the PR8 virus at an MOI 5 for the indicated periods. The total RNA was isolated and analyzed by specific RT-qPCR targeting NP, NEP and NS1 mRNA, which normalized to GAPDH mRNA. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (two-way ANOVA; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001). (C) A549 cells were infected with the indicated virus at an MOI of 5, the total RNA was isolated and analyzed by specific RT-qPCR targeting NEP and NS1 mRNA. Ratios of NEP/NS1 mRNA are presented. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (two-way ANOVA; *, P<0.05). (D-E) (D)A549 cells and (E) MDCK cells were infected with either the wild type or mutant of the PR8 virus at an MOI 0.01 for the indicated periods. The growth curves were determined by TCID50 analysis on MDCK cells. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (two-way ANOVA; ns, not significant; **, P<0.01; ***, P<0.001). (F) A549 cells were infected with the wild type (WT) and mutant (NSA530C) PR8 virus at an MOI of 5. The total RNAs were measured by RT-qPCR and the NEP/NS1 mRNA ratios were calculated. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; ns, not significant; **, P<0.01; ***, P<0.001). (G) A549 cells were infected with the wild type (WT) and mutant (NSA530C) PR8 virus at an MOI of 0.01. The virus titers were detected at 24 hpi. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (one-way ANOVA; ns, not significant; **, P<0.01; ***, P<0.001).
Fig 6.
NSA530C mutation inhibited virus virulence.
(A) MLD50 of WT and mutant viruses. MLD50 was calculated by the Reed and Muench method. (B and C) Weight loss and mortality of mice infected with each indicted virus. The body weight of the WT and mutant groups was compared and statistically analyzed. Error bars represent means ± SEM (n = 10). Statistical analysis of (C) was performed by using Log-rank (Mantel Cox) test. (D) Virus titers in the lungs of infected mice (n = 3) at 3 days (left) and 5 days (right) post-infection. Error bars represent means ± SEM. Statistical analysis was performed by using the one-way ANOVA method (n = 3, ns, not significant; *, P<0.05). (E) Pathological lesions in the lungs of mice infected with the indicated virus at 3 days post-infection with hematoxylin and eosin (H&E) staining. Scale bars, 100 μm. (F) Immunofluorescent staining of lung sections of mice infected with the indicated virus at 3 dpi. The viral NP antigen was stained green, and the nucleus was stained blue. Scale bars, 1000 μm.
Fig 7.
Working model for the regulation of NS mRNA splicing.
NEP mRNA is a spliced form of NS1 mRNA. The splicing complex binds (scissors) to the 3’ splicing site of NS1 mRNA and regulates alternative splicing. YTHDC1 binds to the m6A modification site on the 3’ splice site and inhibits the splicing process. The A530C mutation results in reduced binding of NS mRNA by YTHDC1, which promotes NS1 mRNA splicing and produces more NEP, thereby inhibiting viral polymerase activity.