Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: EBV infection suppresses TLR9 m 6 A modification and expression. A , schematic diagram of the MeRIP-seq protocol used to identify RNA m 6 A profile changes in BJAB cells upon EBV infection. B , GO and KEGG pathway enrichment results for downregulated m 6 A-modified genes upon EBV infection ( p ≤ 0.05, date from our previous study: #GSE133936). C , BJAB cells were infected with 50 MOI EBV for 48 h. TLR9, EBNA1, and GAPDH protein expressions were examined. D , m 6 A -RIP-qPCR analysis of TLR9 mRNA expression in BJAB cells upon EBV infection. Control group was used for negative control. E and F , Flag-TLR9 was pretransfected into HEK293 cells for 6 h, then, Flag-NC, Flag-EBNA1, or Flag-EBNA1△NLS plasmids were respectively transfected for 24 h. The mRNA and protein expressions were measured by RT-qPCR and Western blotting ( E ). The m 6 A abundance of TLR9 mRNA was analyzed by m 6 A -RIP-qPCR (F). Three independent experiments were performed, and data are shown as the mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. EBNA1, Epstein-Barr nuclear antigen 1; EBV, Epstein-Barr virus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; m 6 A, N 6 -methyladenosine; MeRIP, methylated RNA immunoprecipitation; MOI, multiplicity of infection; TLR, toll-like receptor; RIP, RNA immunoprecipitation.

Article Snippet: The antibodies used in this study were TLR9 (13674S, CST), EBNA1 (BM1083, OriGene), GAPDH (60004-1-Ig, Proteintech), METTL3 (15073-1-AP, Proteintech), m 6 A (202003, Synaptic Systems), SALL1 (AWA56088, Abiowell),PRKN(AWA10174, Abiowell), MID1 (AWA40807, Abiowell), Myc-tag(60003-2-Ig, Proteintech), TLR3 (AWA50624, Abiowell), TLR7 (AWA58104, Abiowell), TLR8 (AWA58112, Abiowell), ALKBH5 (ab69325, Abcam), FTO (27226-1-AP, Proteintech), GFP (YM3124, Immunoway), α-Tubulin (66031-1-Ig, Proteintech), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (25537-1-AP, Proteintech), YTHDC1 (14392-1-AP, Proteintech), TBK1 (38066, CST), p-TBK1 (5483, CST), MYD88 (4283, CST), p-P65 (S536) (3033S, CST), p65 (8242, CST), IRF3 (11904, CST), Histone H3 (A2348, Abclonal), ubiquitin (20326, CST), Goat anti-Rabbit IgG H&L (HRP) (511203, ZEN-Bioscience), Goat anti-Mouse IgG H&L (HRP) (511103, ZEN-Bioscience).

Techniques: Infection, Modification, Expressing, Control, Negative Control, Transfection, Quantitative RT-PCR, Western Blot, Virus, Methylation, RNA Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: TLR9 downstream signaling pathways and cytokine expression are regulated by METTL3. A , cellular m 6 A modification levels were assayed in BJAB-shNC and BJAB-shMETTL3 cells by Dot blot analysis. B , the expression levels of TLR9 and METTL3 were determined by Western blotting in BJAB-shNC and BJAB-shMETTL3 cells. C , BJAB-shNC and BJAB-shMETTL3 cells were treated with ActD (5 μg/ml) for 0, 3, and 6 h; TLR9 mRNA levels were measured by RT-qPCR. D , the indicated TLR9 signaling proteins were detected by cytoplasmic and nuclear proteins isolation. GAPDH and Histone H3 were used for cytoplasm and nucleus protein loading controls, respectively. E , BJAB cells were treated with STM2457(10 μM) and CpG-ODN2006 (5 μM) upon 50 MOI EBV infection, respectively. EBV genomes copy number was determined by qPCR. F , primary B cells were treated with STM2457(10 μM) and CpG-ODN2006(5 μM) with or without 50MOI EBV infection for appointed time. The secretion of IFN-α and CXCL10 were assayed by ELISA. These data are shown as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. EBNA1, Epstein-Barr nuclear antigen 1; EBV, Epstein-Barr virus; IFN, interferon; m 6 A, N 6 -methyladenosine; MOI, multiplicity of infection; ODN, oligodeoxynucleotide; RT-qPCR, reverse transcription-quantitative PCR; TLR9, toll-like receptor 9.

Article Snippet: The antibodies used in this study were TLR9 (13674S, CST), EBNA1 (BM1083, OriGene), GAPDH (60004-1-Ig, Proteintech), METTL3 (15073-1-AP, Proteintech), m 6 A (202003, Synaptic Systems), SALL1 (AWA56088, Abiowell),PRKN(AWA10174, Abiowell), MID1 (AWA40807, Abiowell), Myc-tag(60003-2-Ig, Proteintech), TLR3 (AWA50624, Abiowell), TLR7 (AWA58104, Abiowell), TLR8 (AWA58112, Abiowell), ALKBH5 (ab69325, Abcam), FTO (27226-1-AP, Proteintech), GFP (YM3124, Immunoway), α-Tubulin (66031-1-Ig, Proteintech), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (25537-1-AP, Proteintech), YTHDC1 (14392-1-AP, Proteintech), TBK1 (38066, CST), p-TBK1 (5483, CST), MYD88 (4283, CST), p-P65 (S536) (3033S, CST), p65 (8242, CST), IRF3 (11904, CST), Histone H3 (A2348, Abclonal), ubiquitin (20326, CST), Goat anti-Rabbit IgG H&L (HRP) (511203, ZEN-Bioscience), Goat anti-Mouse IgG H&L (HRP) (511103, ZEN-Bioscience).

Techniques: Protein-Protein interactions, Expressing, Modification, Dot Blot, Western Blot, Quantitative RT-PCR, Isolation, Infection, Enzyme-linked Immunosorbent Assay, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: EBNA1 induces METTL3 protein degradation via the ubiquitination pathway. A , EBNA1 protein levels were detected in BJAB, Raji, and B95.8 cells. B and C , Flag-NC and Flag-EBNA1 were transfected into HEK293 cells. The mRNA and protein levels of METTL3 and EBNA1 were assayed by RT-qPCR ( B ) and Western blotting ( C ). D , The 100 ng, 200 ng, and 500 ng mRNA from HEK293 cells transfected with Flag-EBNA1 (or Flag-NC as negative control) were extracted to detect the total cellular m 6 A modification level by Dot blot. A concentration of 0.02% methylene blue staining was used as a loading control. E and F , HEK293 cells were transfected with EBNA1 plasmids for 36 h, followed by CHX treatment (25 μg/ml) and MG132 (25 μM) for indicated times. Then cellular proteins were collected, and Western blotting was performed. G , GFP-NC and GFP-EBNA1, Flag-METTL3 and plasmids containing various polyubiquitin chains were cotransfected for 48 h. HEK293 cells were treated with MG132 (25 μM) for 4 h before harvest. Lysates were immunoprecipitated with anti-Flag beads. H , HONE-1 cells were transfected with indicated siRNAs for 48 h. The protein expression levels were detected by Western blotting. I and J , HONE-1 cells were transfected with indicated plasmids, then, the mRNA and protein expression of PRKN was measured by RT-qPCR ( I ) and Western blotting ( J ). K , HEK293 cells were transfected with Flag-PRKN and Myc-METTL3 plasmids for 48 h. The cell lysates were immunoprecipitated with anti-Flag beads (or IgG as control). The first lane was an input control without immunoprecipitation. L , Myc-METTL3, Flag-PRNK, and Ub (either WT or K48R) plasmids were cotransfected into HEK293 cells for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibodies. The data are shown as the mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. CHX, cycloheximide; EBNA1, Epstein-Barr nuclear antigen 1; m 6 A, N 6 -methyladenosine; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: The antibodies used in this study were TLR9 (13674S, CST), EBNA1 (BM1083, OriGene), GAPDH (60004-1-Ig, Proteintech), METTL3 (15073-1-AP, Proteintech), m 6 A (202003, Synaptic Systems), SALL1 (AWA56088, Abiowell),PRKN(AWA10174, Abiowell), MID1 (AWA40807, Abiowell), Myc-tag(60003-2-Ig, Proteintech), TLR3 (AWA50624, Abiowell), TLR7 (AWA58104, Abiowell), TLR8 (AWA58112, Abiowell), ALKBH5 (ab69325, Abcam), FTO (27226-1-AP, Proteintech), GFP (YM3124, Immunoway), α-Tubulin (66031-1-Ig, Proteintech), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (25537-1-AP, Proteintech), YTHDC1 (14392-1-AP, Proteintech), TBK1 (38066, CST), p-TBK1 (5483, CST), MYD88 (4283, CST), p-P65 (S536) (3033S, CST), p65 (8242, CST), IRF3 (11904, CST), Histone H3 (A2348, Abclonal), ubiquitin (20326, CST), Goat anti-Rabbit IgG H&L (HRP) (511203, ZEN-Bioscience), Goat anti-Mouse IgG H&L (HRP) (511103, ZEN-Bioscience).

Techniques: Ubiquitin Proteomics, Transfection, Quantitative RT-PCR, Western Blot, Negative Control, Modification, Dot Blot, Concentration Assay, Staining, Control, Immunoprecipitation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: A working model depicting the main molecular mechanisms about the current study. EBV EBNA1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway, which mediated by the E3 ligase PRKN, thus inhibits cellular m 6 A modification. Downregulation of METTL3 inhibits TLR9 m 6 A modification. YTHDF1 is an m 6 A “reader” for cellular TLR9 mRNA with m 6 A modification, and YTHDF1 binding increases TLR9 mRNA translation efficiency. EBV infection decreases the m 6 A levels of TLR9 mRNA, thus reduces TLR9 protein levels, as well as TLR9-induced cytokine secretions. EBV, Epstein-Barr virus; m 6 A, N 6 -methyladenosine; TLR9, toll-like receptor 9.

Article Snippet: The antibodies used in this study were TLR9 (13674S, CST), EBNA1 (BM1083, OriGene), GAPDH (60004-1-Ig, Proteintech), METTL3 (15073-1-AP, Proteintech), m 6 A (202003, Synaptic Systems), SALL1 (AWA56088, Abiowell),PRKN(AWA10174, Abiowell), MID1 (AWA40807, Abiowell), Myc-tag(60003-2-Ig, Proteintech), TLR3 (AWA50624, Abiowell), TLR7 (AWA58104, Abiowell), TLR8 (AWA58112, Abiowell), ALKBH5 (ab69325, Abcam), FTO (27226-1-AP, Proteintech), GFP (YM3124, Immunoway), α-Tubulin (66031-1-Ig, Proteintech), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (25537-1-AP, Proteintech), YTHDC1 (14392-1-AP, Proteintech), TBK1 (38066, CST), p-TBK1 (5483, CST), MYD88 (4283, CST), p-P65 (S536) (3033S, CST), p65 (8242, CST), IRF3 (11904, CST), Histone H3 (A2348, Abclonal), ubiquitin (20326, CST), Goat anti-Rabbit IgG H&L (HRP) (511203, ZEN-Bioscience), Goat anti-Mouse IgG H&L (HRP) (511103, ZEN-Bioscience).

Techniques: Ubiquitin Proteomics, Modification, Binding Assay, Infection, Virus