Fig 1.
TET2 expression is reduced in H9N2 JSC1 infected THP-1 cells and A549 cells.
THP-1 cells (A) or A549 cells (C) were infected with JSC1 at indicated MOIs and harvested at 9 hours post-infection (hpi). THP-1 cells (B and E) or A549 cells (D and F) were infected with JSC1 at an MOI of 1 and harvested at 0, 6, 12 and 24 hpi. (A-D) Protein levels of TET2, viral NP, and GAPDH were analyzed by Western Blot. (E and F) TET2 mRNA and 18S rRNA levels were detected by RT-qPCR. The data are expressed as fold changes in TET2 mRNA levels at 6, 12, and 24 hpi relative to 0 hpi control. (G) THP-1 cells or A549 cells were infected with JSC1 at an MOI of 1 and harvested at 12 hpi. 18S rRNA, TET2 mRNA and TET2 pre-mRNA levels were detected by RT-qPCR. The data are expressed as fold changes relative to the mock infections. Error bars represent ± SD for triplicate experiments. Statistical analysis was performed using Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2.
PA-X targets TET2 mRNA for degradation with dependence on the endoribonuclease activity.
(A, B and D) HEK293T cells were transfected with the indicated plasmids, and whole-cell extracts were prepared at 24 hours post-transfection (hpt), followed by immunoblotting with indicated antibodies. (C) Diagrams of expressing proteins in indicated plasmids are shown. (E) HEK293T cells were transfected with the indicated plasmids and harvested at 24 hpt. TET2 mRNA and 18S rRNA levels were detected by RT-qPCR. The data are expressed as fold changes relative to the FLAG-empty control. (F) RNA-binding protein immunoprecipitation (RIP) was performed with anti-FLAG M2 agarose beads in HEK293T cells transfected with FLAG-PA-X D108A, FLAG-PA-N D108A or FLAG-empty for 24 hours, followed by immunoblotting to verify the efficiency of immunoprecipitation, and by RT-qPCR to measure the TET2 mRNA and 18S rRNA levels. RNA enrichment was calculated by normalization of each IP fractions’ Ct to the input fraction’ Ct in the same qPCR assay (ΔCt). (G) RNA and protein samples were collected from THP-1 cells expressing doxycycline-inducible FLAG-PA-X, FLAG-PA-X D108A, or EGFP 18 hours after the supplement of doxycycline, followed by immunoblotting analysis and RT-qPCR detection. The RT-qPCR data are expressed as fold changes relative to the EGFP control. (H and I) Protein and RNA samples were collected from THP-1 cells infected with either PR8 WT or PR8 ΔPA-X at an MOI of 1, followed by immunoblotting analysis (H) and RT-qPCR detection (I). Error bars represent ± SD for triplicate experiments. Statistical analysis was performed using Student’s t test or ANOVA method. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3.
Loss of TET2 enhances IAV replication.
CRISPR-Cas9-mediated TET2 knockout (KO) and non-targeting gRNA control (CT) THP-1 cells were established. (A) DNA sequencing results of sgRNA-targeted TET2 genomic region in two isolated TET2-KO single cell clones (KO-1 and KO-2) and CT THP-1 cells are shown. Hyphen (-) represents missing DNA bases, and asterisk (*) indicates the termination codon (TAA). (B) Western Blot confirmation of TET2 knockout in THP-1 cells. (C) CT, KO-1 and KO-2 THP-1 cells were seeded in the 96-well plate at equal amount cells (8 × 103). Cell numbers were detected at indicated hours using Cell Counting Kit-8 (CCK-8) reagents. (D and E) CT, KO-1 and KO-2 THP-1 cells were infected with H9N2 JSC1 at an MOI of 1 and harvested at 6 hpi. (D) Protein levels of TET2, viral NP, viral NS1 and GAPDH were analyzed by Western Blot. (E) mRNA levels of viral NS1 were detected by RT-qPCR, and the data are expressed as fold changes relative to the control group. (F) Control and TET2-KO THP-1 cells were infected with JSC1 at an MOI of 1 and harvested at 0, 3, 6, 12 and 24 hours, followed by immunoblotting with indicated antibodies. (G) Control and TET2-KO THP-1 cells were infected with JSC1 at an MOI of 1, and the culture supernatants were collected at 6, 12 and 24 hpi for viral titration by TCID50 assay. (H) Control and TET2-KO THP-1 cells were infected with H9N2 JSC1, H9N2 Ch01, H1N1 PR8 or H3N2 ZJ163 at an MOI of 1 and harvested at 6 hpi, followed by immunoblotting with indicated antibodies. (I) Control and TET2-KO THP-1 cells transduced with lentiviral vectors expressing EGFP or FLAG-TET2 CD domain were infected with JSC1 at an MOI of 1 and harvested at 6 hpi, followed by immunoblotting with indicated antibodies. (J) Control and TET2-KO THP-1 cells were infected with either PR8 WT or PR8 ΔPA-X at an MOI of 1 and harvested at 6 hpi, followed by immunoblotting with indicated antibodies. Error bars represent ± SD for triplicate experiments. Statistical analysis was performed using Student’s t test or ANOVA method. *p < 0.05, **p < 0.01, ***p < 0.001. CT: control cells, KO: TET2 knockout cells.
Fig 4.
TET2 deletion attenuates the demethylation and transcription of STAT1 and ISGs.
(A) Control and TET2-KO THP-1 cells were infected with JSC1 at an MOI of 1. At 6 hpi, total RNA was extracted from cells, and RIG-1, TLR3, TLR7, IRF3, IRF7 and IFNB1 mRNA levels (upstream of IFN) and STAT1, ISG15, ISG20, IFITM3, OAS1, IFIT5, MOV10 and TRIM25 mRNA levels (downstream of IFN) were evaluated via RT-qPCR. The data are expressed as fold changes relative to the control group. (B) Control and TET2-KO THP-1 cells were treated with indicated amounts of rHuIFN-βb1 for 2 hours and then infected with JSC1 at an MOI of 1 and harvested at 6 hpi, followed by immunoblotting with indicated antibodies. (C-E) Hydroxymethylated DNA immunoprecipitation and sequencing (hMeDIP-Seq) was performed in control and TET2-KO THP-1 cells infected with JSC1 at an MOI of 3 for 6 hours. (C) Peaks were identified using the MACS algorithm (p-value = 1e-5). The bar chart displays the numbers of total peaks. (D) Distribution of signal value are shown, which indicates the frequency of peaks with different fold enrichments. (E) Distribution of 5hmC along promoter regions of STAT1 (chr2 191,876,601–191,882,599), ISG15 (chr1 945,390–951,388), ISG20 (chr15 89,175,708–89,181,706) and IFIT5 (chr10 91,170,072–91,176,070) genes in control (blue) and TET2-KO (red) cells are shown. The regions of differential 5hmC levels are highlighted by black squares. (F) hMeDIP assays were performed in control and TET2-KO THP-1 cells with or without JSC1 infection. 5hmC levels on the STAT1, ISG15, ISG20 and IFIT5 promoters were determined by qPCR. 5hmC enrichment was calculated by normalization of each IP fractions’ Ct to the input fraction’ Ct in the same qPCR assay (ΔCt). (G) Control and TET2-KO THP-1 cells were infected with either PR8 WT or PR8 ΔPA-X at an MOI of 1 and harvested at 6 hpi, and mRNA levels of STAT1, ISG15, ISG20 and IFIT5 were evaluated via RT-qPCR. Error bars represent ± SD for triplicate experiments. Statistical analysis was performed using Student’s t test or ANOVA method. *p < 0.05, **p < 0.01, ***p < 0.001. CT: control cells, KO: TET2 knockout cells.
Fig 5.
TET2 regulates STAT1 and ISG expression.
(A) Dual-luciferase reporter assays were performed in HEK293T cells transfected with plasmids expression FLAG-TET2 and FLAG-empty proteins. Firefly luciferase activity driven by STAT1 promoter was measured at 24 hpt and normalized to Renilla luciferase activity. Error bars were calculated from three technical replicates. (B) Control THP-1 cells ectopically expressing EGFP, and TET2-KO THP-1 cells ectopically expressing EGFP or FLAG-TET2 CD domain were infected with JSC1 at an MOI of 1, and STAT1 mRNA levels were evaluated at 6 hpi via RT-qPCR. The data are expressed as fold changes relative to the mock EGFP-control group. (C) Control and TET2-KO THP-1 cells were infected with JSC1 at an MOI of 1 and harvested at 0, 3, 6, 12 and 24 hpi, followed by immunoblotting with indicated antibodies. (D) Control and TET2-KO THP-1 cells transduced with lentiviral vectors expressing EGFP or STAT1 were infected with JSC1 at an MOI of 1 and harvested at 6 hpi, followed by immunoblotting with indicated antibodies. (E) Control THP-1 cells ectopically expressing EGFP and TET2-KO THP-1 cells ectopically expressing EGFP, FLAG-TET2 CD domain or STAT1 were treated with 100 U/mL recombinant rHuIFN-βb1 and harvested at 3 hpi. The mRNA levels of ISG15, ISG20 and IFIT5 were evaluated via RT-qPCR. The data are expressed as fold changes relative to the mock EGFP-control group. Error bars represent ± SD for triplicate experiments. Statistical analysis was performed using Student’s t test or ANOVA method. *p < 0.05, **p < 0.01, ***p < 0.001. CT: control cells, KO: TET2 knockout cells.
Fig 6.
TET2 deletion enhances influenza infection and replication in mice.
4-week-old wild-type (Tet2+/+), heterozygous Tet2-KO (Tet2+/-) and homozygous Tet2-KO (Tet2-/-) C57BL/6 mice were intranasally infected with H9N2 Ch01 (104.5 TCID50) and monitored every 24 h for mortality and morbidity. (A) Kaplan-Meier survival curves within 13 dpi are shown (n = 9/group). Statistical analysis was performed using log-rank (Mantel-Cox) test. *p < 0.05. Nasal lavage fluid (B) and lung grinding supernatant (C) of mice sacrificed on 4 dpi were collected for viral titration by TCID50 assay (n = 5/group). Error bars represent ± SD. Statistical analysis was performed using ANOVA method. *p < 0.05, **p < 0.01. (D) Histopathology of lungs of mice sacrificed on 7 dpi are shown (n = 1/group of mock treatment, n = 2/group of infected treatment). Scale bars, 200 μm.