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