Journal: Immunity

Article Title: The pre-exposure SARS-CoV-2-specific T cell repertoire determines the quality of the immune response to vaccination

doi: 10.1016/j.immuni.2022.08.003

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Article Snippet: PepTivator EBV EBNA-1 , Miltenyi Biotec , Cat#130-093-613.

Techniques: Functional Assay, Recombinant, Staining, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Software

Journal: mBio

Article Title: EBNA1 SUMOylation by PIAS1 suppresses EBV lytic replication and enhances episome maintenance

doi: 10.1128/mbio.02639-25

Figure Lengend Snippet: PIAS1 interacts with EBNA1. ( A and B ) HEK-293T ( A ) and HEK-293 (EBV+) ( B ) cells were co-transfected with PIAS1 and V5-EBNA1 as specified. WB analysis shows that PIAS1 is co-IPed with EBNA1. Whole-cell lysates were probed for PIAS1 and V5-EBNA1 to confirm input levels. β-actin was used as a loading control. ( C and D ) Akata (EBV+) ( C ) and SNU-719 ( D ) cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature. Subsequently, the cells were incubated with either PBS control or a combination of mouse anti-EBNA1 and rabbit anti-PIAS1 antibodies. Probes were then added for ligation and amplification. Cell nuclei were visualized using Nikon AXR after staining with 4′,6-diamidino-2-phenylindole (DAPI). The interaction between EBNA1 and PIAS1 in situ was indicated by red dots representing proximity ligation assay (PLA) signals.

Article Snippet: Briefly, cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature for 1 h, then incubated with PBS control or a mixture of mouse anti-EBNA1 (Cat. #sc-81581, Santa Cruz) and rabbit anti-PIAS1 (Cat. #ab77231, Abcam) or rabbit anti-SUMO2/3 (Cat. # 11251-1-AP, Proteintech) antibodies (1:50 dilution in 3% BSA) at 4°C overnight.

Techniques: Transfection, Control, Saline, Incubation, Ligation, Amplification, Staining, In Situ, Proximity Ligation Assay

Journal: mBio

Article Title: EBNA1 SUMOylation by PIAS1 suppresses EBV lytic replication and enhances episome maintenance

doi: 10.1128/mbio.02639-25

Figure Lengend Snippet: PIAS1 enhances EBNA1 SUMOylation both in vivo and in vitro . ( A ) HEK-293T cells were transfected with plasmids encoding Halo-V5-EBNA1, Halo-PIAS1, and His-SUMO2. Whole-cell lysates (input) were analyzed by WB using antibodies against SUMO2/3, PIAS1, V5, and β-actin. SUMOylated proteins are indicated by brackets. EBNA1 was immunoprecipitated using anti-V5 magnetic beads, followed by WB analysis with antibodies as indicated. Arrows denote SUMOylated EBNA1. ( B ) In vitro SUMOylation assay was conducted using a combination of purified proteins, including E1, E2, SUMO2, PIAS1, and the substrate V5-EBNA1, as specified. The reaction was stopped by adding 2× SDS-PAGE loading buffer, followed by WB analysis using anti-V5-HRP antibody. SUMOylated EBNA1 is indicated by brackets. ( C ) WB analysis of PIAS1 and β-actin expression in non-targeting control (NC) and PIAS1-depleted (sg-PIAS1) Akata (EBV+) cells. ( D ) Control (NC) and PIAS1-depleted (sg-PIAS1) Akata (EBV+) cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature, followed by incubation with mouse anti-EBNA1 and rabbit anti-SUMO2/3 antibodies. PLA probes were subsequently added for ligation and amplification. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and visualized using a Nikon AXR confocal microscope. The close proximity between EBNA1 and SUMO2/3 is indicated by red fluorescent PLA signals.

Article Snippet: Briefly, cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature for 1 h, then incubated with PBS control or a mixture of mouse anti-EBNA1 (Cat. #sc-81581, Santa Cruz) and rabbit anti-PIAS1 (Cat. #ab77231, Abcam) or rabbit anti-SUMO2/3 (Cat. # 11251-1-AP, Proteintech) antibodies (1:50 dilution in 3% BSA) at 4°C overnight.

Techniques: In Vivo, In Vitro, Transfection, Immunoprecipitation, Magnetic Beads, Purification, SDS Page, Expressing, Control, Saline, Incubation, Ligation, Amplification, Staining, Microscopy

Journal: mBio

Article Title: EBNA1 SUMOylation by PIAS1 suppresses EBV lytic replication and enhances episome maintenance

doi: 10.1128/mbio.02639-25

Figure Lengend Snippet: SUMOylation-deficient EBNA1 facilitates EBV replicon loss. ( A ) Schematic representation of EBV replicon retention assays using HEK-293T cells carrying WT EBNA1 and RRR mutant plasmids. ( B ) WB analysis comparing EBNA1 and β-actin expression in HEK-293T cells carrying pCEP4-EBNA1-WT and pCEP4-EBNA1-RRR plasmids at 0 and 9 days post-hygromycin removal. ( C ) The remaining EBV replicon was measured by qPCR over 9 days after removal of hygromycin B. ( D ) ChIP-qPCR analysis of EBNA1 binding to oriP in cells carrying pCEP4-EBNA1 WT and RRR plasmids. Anti-EBNA1 antibody was used for EBNA1 ChIP, and nonspecific IgG was used as a negative control. Data represent ± SD from three biological replicates. ** P < 0.01; *** P < 0.001.

Article Snippet: Briefly, cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature for 1 h, then incubated with PBS control or a mixture of mouse anti-EBNA1 (Cat. #sc-81581, Santa Cruz) and rabbit anti-PIAS1 (Cat. #ab77231, Abcam) or rabbit anti-SUMO2/3 (Cat. # 11251-1-AP, Proteintech) antibodies (1:50 dilution in 3% BSA) at 4°C overnight.

Techniques: Mutagenesis, Expressing, ChIP-qPCR, Binding Assay, Negative Control

Journal: BMC Cancer

Article Title: miR-18a reactivates the Epstein-Barr virus through defective DNA damage response and promotes genomic instability in EBV-associated lymphomas

doi: 10.1186/s12885-018-5205-9

Figure Lengend Snippet: Expression of miR-18a in lymphoma patients. a Relative expression of miR-18a in diffuse large B-cell lymphoma patients and normal controls; publicly available microRNA array data (GSE42906) were compared between groups with GEO2R. b Unsupervised hierarchical clustering of microRNA expression. The miR-17-92 cluster and EBV-encoded microRNAs were differentially expressed between EBV- positive and -negative B cells; High and low expression levels are indicated by red and green, respectively. The raw data are shown in NCBI, GEO:GSE36926. c Expression of miR-18a and EBNA1. The expression of miR-18a was measured by in situ hybridization. The expression of EBNA1 was measured by immunohistochemistry. Representative figures are shown (× 100); Upper left and upper right: lymphoma biopsies; lower left and lower right: normal control lymph nodes. d Scatter plot of the observed expression scores of miR-18a. Expression was scored semi-quantitatively by multiplying the intensity and area of staining. e Correlation of the expression of EBNA1 and miR-18a. f Kaplan-Meier curves for patients according to the tumor expression of miR-18a. g Kaplan-Meier curves for patients according to the tumor expression of EBNA1

Article Snippet: The antibodies were used as follows: ATM, EBNA1 (Novus Biologicals, USA), γ-H2AX(Abcam, USA), β-actin (Santa Cruz Biotechnology, CA, USA) and GAPDH (cell signaling, USA).

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Control, Staining

Journal: BMC Cancer

Article Title: miR-18a reactivates the Epstein-Barr virus through defective DNA damage response and promotes genomic instability in EBV-associated lymphomas

doi: 10.1186/s12885-018-5205-9

Figure Lengend Snippet: miR-18a increases the EBV viral load. a Transfection of miR-18a mimics in P3HR-1 and Raji resulted in a higher EBV viral load. Quantitative real-time EBV PCR was performed in samples collected from the culture media of cells. Viral DNA was extracted, and PCR was carried out according to the instructions. The EBV copy number was calculated according to a standard curve. b Expression of EBNA1 after transfection of miR-18a mimics and inhibitor in Raji cells as measured by western blotting. c Relative expression of EBV gene expression after transfection of miR-18a. d Visualization of episomal and integrated EBV DNA by fluorescence in situ hybridization

Article Snippet: The antibodies were used as follows: ATM, EBNA1 (Novus Biologicals, USA), γ-H2AX(Abcam, USA), β-actin (Santa Cruz Biotechnology, CA, USA) and GAPDH (cell signaling, USA).

Techniques: Transfection, Expressing, Western Blot, Gene Expression, Fluorescence, In Situ Hybridization

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