Fig 1: SMC6 associates with EBV genomic R-loop regions in the absence of BNRF1(A) Confocal immunofluorescence analysis of P3HR-1 ZHT/RHT cells stained with S9.6, anti-SMC6-HA, or DAPI. S9.6 recognizes R-loops and double-stranded RNA (dsRNA). Cells were not induced for exogenous SMC6-HA (-dox) or for lytic reactivation (-4-HT/NaB). Representative of 3 independent experiments.(B) Confocal analysis of P3HR-1 ZHT/RHT cells treated with dox (5 µg/mL) to induce exogenous SMC6 expression and with 4-HT/NaB for 9 h to induce EBV lytic reactivation, as indicated. Cells were then stained with S9.6, anti-HA-SMC6, or DAPI. Representative of 3 independent experiments.(C) Confocal analysis of cells as in (B) that were pre-incubated with RNaseH (5.5 U/µg DNA) prior to staining with S9.6, anti-HA, or DAPI. Representative of 3 independent experiments.(D) Confocal analysis of cells as in (B) that were stained with the anti-dsRNA antibody rJ2 or DAPI. Representative of 3 independent experiments.(E) Confocal analysis of cells as in (B) that were pre-incubated with RNaseH (5.5 U/µg DNA) prior to staining with the anti-dsRNA antibody rJ2 or DAPI. Representative of 3 independent experiments.(F) Confocal analysis of cells as in (B) that were stained with S9.6, anti-F-actin, or DAPI. Representative of 3 independent experiments.(G) Confocal analysis of cells as in (B) that were stained with the anti-dsRNA antibody rJ2, anti-HA-SMC6, anti-F-actin, or DAPI, as indicated. Representative of 3 independent experiments.(H) Confocal analysis of cells as in (B) that were pre-incubated with RNaseH (5.5 U/µg DNA) prior to staining with the anti-dsRNA antibody rJ2, anti-HA-SMC6, anti-F-actin, or DAPI. Representative of 3 independent experiments.(I) Immunoblot analysis of 1% input and S9.6-immunopurified complexes from P3HR-1 ZHT/RHT cells induced for exogenous HA-SMC6 and treated with 4-HT/NaB and phosphonoacetic acid (PAA; 200 µg/mL) for 9 h, as indicated. Samples were treated with RNaseH (5.5 U/µg DNA) or benzonase (5 U/µg DNA) prior to immunoprecipitation (IP), as indicated.(J) Mean ± SEM values from 3 replicates of ChIP-qPCR analysis of SMC6 occupancy at the EBV genomic oriLytR region. Anti-HA ChIP was performed on chromatin from EBV+ Akata cells with dox induced (5 µg/mL) SMC6-HA or GFP-HA expression following 48 h of aIgG crosslinking, followed by qPCR using oriLytR region-specific primers. *p < 0.05.
Fig 2: BNRF1 is critical for viral RC formation(A) Immunofluorescence analysis of replication compartments (RCs), as judged by EdC-labeled DNA and staining for BMRF1 in P3HR-1 ZHT/RHT cells that expressed the indicated sgRNAs and that were induced by 4-HT/NaB for 24 h. Magnified images of cells in white boxes are shown at the bottom.(B) 3D reconstruction of EdC and BMRF1 signals as in (A), using the ImageJ Interactive 3D Surface Plot package. The scale bar indicates fluorescence intensity.(C) Mean ± SEM percentages of nuclei with RCs from 3 replicates as in (A), using data from 10 randomly selected panels of 150 nuclei and ImageJ.(D) RC immunofluorescence analysis as in (A), using P3HR-1 ZHT/RHT cells that expressed the indicated sgRNAs and rescue BNRF1 cDNA.(E) 3D reconstruction of EdC and BMRF1 signals as in (D), using the ImageJ Interactive 3D Surface Plot package.(F) Mean ± SEM percentages of nuclei with RCs from 3 replicates as in (D), using data from 5 randomly selected panels of 75 nuclei and ImageJ.(G) RC immunofluorescence analysis as in (A), using P3HR-1 ZHT/RHT cells that expressed no sgRNA, control or SMC6 sgRNA, and were treated with bortezomib, as indicated.(H) 3D reconstruction of EdC and BMRF1 signals as in (G), using the ImageJ Interactive 3D Surface Plot package.(I) Mean ± SEM percentages of nuclei with RCs from 3 replicates as in (G), using data from 5 randomly selected panels of 75 nuclei and ImageJ.(J) RC immunofluorescence analysis as in (A), using P3HR-1 ZHT/RHT cells that expressed the indicated sgRNAs.(K) 3D reconstruction of EdC and BMRF1 signals as in (J), using the ImageJ Interactive 3D Surface Plot package.(L) Mean ± SEM percentages of nuclei with RCs from 3 replicates as in (D), using data from 4 randomly selected panels of 60 nuclei and ImageJ.See also Figures S5-S7.
Fig 3: BNRF1 associates with SMC6(A) Immunoblot analysis of 1% input and anti-HA immunopurified complexes from P3HR-1 ZHT/RHT left untreated or induced for GFP versus BNRF1 cDNAs with dox (5 µg/mL) for 6 h and treated with bortezomib (5 nM) for 6 h. Representative of two independent experiments.(B) Immunofluorescence analysis of dox-induced (5 µg/mL) HA-tagged SMC6 and V5-tagged BNRF1 versus nuclear DAPI signals in EBV+ Akata cells treated with bortezomib (5 nM) and MLN4924 (10 µM) for 12 h. Scale bar, 2 µm. Representative of 3 experiments.(C) Schematic of BNRF1 wild-type (WT) and deletion mutant constructs used.(D) Immunoblot analysis of WCL from P3HR-1 ZHT/RHT cells left untreated or induced for BNRF1 cDNA expression by dox (5 µg/mL) for 24 h. Representative of 2 replicates.(E) Immunofluorescence analysis of dox-induced (5 µg/mL) HA-tagged BNRF1 expression for 24 h versus DAPI staining in P3HR-1 ZHT/RHT cells. Magnified images of cells boxed in white are shown in the top two rows. Representative of 3 experiments.See also Figure S3.
Fig 4: The SMC5/6 cohesin complex is depleted by incoming EBV and lytic replication(A) Waterfall plot illustrating the log2 fold change in protein abundance of host proteins that significantly change (p < 0.05) 24 h after lytic reactivation of P3HR-1 ZHT/RHT cells mock-induced or induced for lytic reactivation by 4-HT (Ersing et al., 2017).(B) Schematic of the SMC5/6 complex.(C) Temporal proteomic plots of SMC5/6 complex subunit relative protein abundances in mock-induced (red) or 4-HT induced ZHT/RHT P3HR-1 cells that entered productive lytic cycles and expressed the late gene gp350 (green), or that did not become gp350+ (orange), are indicated. Error bars indicate mean ± SE (n = 3 replicates).(D)Immunoblot analysis of whole-cell lysates (WCLs) from EBV+ AGS gastric carcinoma cells with a conditional BZLF1 allele induced for lytic replication by doxycycline (2 µg/mL) for 48 h.(E) Plot of -log2 fold change in protein abundance of host proteins that were significantly different in proteomics analysis of primary human B cells infected with B95.8 EBV at 48 h of infection versus in resting B cells.(F) Temporal proteomics plots of SMC5/6 complex abundance on the indicated days after primary human B-cell EBV infection. Data show the mean ± SE (n = 4).(G) Immunoblot of SMC6 versus the DDX46 load control values in primary (top) or Daudi B cells at 0 versus 3 days post infection (DPI).Blots in (D) and (G) are representative of 2 replicates. See also Figure S1.
Fig 5: The EBV tegument protein BNRF1 targets SMC5/6 for proteasomal degradation in a calpain-dependent manner(A) Volcano plot analysis of changes in proteomic abundances in primary human B cells at 2 DPI versus uninfected.(B) Relative BNRF1 protein abundances at the indicated days after primary B cell EBV infection.(C) Immunoblot analysis of WCL from P3HR-1 cells mock induced or doxycycline (dox; 5 µg/mL) induced for EBV lytic gene cDNA expression for 24 h.(D) Immunoblot analysis of WCL from Cas9+ P3HR-1 ZHT/RHT cells expressing the indicated sgRNA and mock induced or induced for lytic reactivation by 4-HT (400 nM) and sodium butyrate (NaB; 500 µM) for 24 h.(E) Immunoblot analysis of P3HR-1 ZHT/RHT WCL from cells mock induced or dox induced for BNRF1 cDNA expression in the presence of bortezomib (5 nM) or MLN4924 (10 µM), as indicated, for 24 h.(F) BNRF1-selective protein interactors identified by affinity purification, HA peptide elution, and mass spectrometry analysis of dox-induced HA-BNRF1 in P3HR-1 ZHT/RHT cells induced for lytic replication by 4-HT/NaB and cross-compared with the HA-tagged tegument protein controls BPLF1, BLRF2 and BOLF1.(G) Immunoblot analysis of 1% input and anti-HA immunopurified GFP or SMC6 complexes from P3HR-1 ZHT/RHT left untreated or treated with dox (5 µg/mL) and bortezomib (5 nM) for 6 h, as indicated. Representative of two independent experiments.(H) Immunoblot analysis of WCL from P3HR-1 ZHT/RHT cells with dox-induced HA-tagged BNRF1 and treated with bortezomib, MLN4924, or calpeptin for 24 h, as indicated.(I) Immunoblot analysis of WCL from P3HR-1 ZHT/RHT cells expressing the indicated sgRNAs and induced for lytic replication, as indicated.Blots are representative of 2 independent replicates unless otherwise indicated. See also Figure S2.
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