EBV⁺ AKBM BL cells were treated with anti-human IgG (αIgG) to induce viral replication. (a) At the indicated times post induction, percentages of productively EBV-infected AKBM cells were determined by flow cytometric analysis of induced ratCD2-GFP reporter expression. (b) In post-nuclear AKBM cell lysates, active DUBs were labeled with a fluorescent Ub-VME probe, resolved by SDS-PAGE, and visualized by in-gel fluorescence imaging. The arrow indicates a band appearing in AKBM cells after 9 hours of lytic cycle induction (lanes 4 and 5). (c) DUB profiles of EBV⁻ AK31-rCD2-GFP cells (AK31, lanes 1 and 2), EBV⁺ AKBM cells (lanes 3 and 4), and transfected 293T cells (lanes 5–9). In parallel with the EBV⁺ AKBM cells, EBV⁻ control AK31 cells were treated with αIgG for 24 hours; this resulted in productive infection in 56% of AKBM cells; AK31 cells included a population of 45% that expressed ratCD2-GFP from a constitutive promoter irrespective of αIgG treatment (data not shown). For comparison, 293T cells were transfected with constructs encoding three (putative) EBV DUBs: BPFL1, BXLF1, BSLF1, or the 325 aa N-terminal part of BPLF1. The asterisk marks a smaller fragment observed upon transfection of 293T cells with full-length or the N-terminal part of BPLF1 (lanes 6 and 9). Left and right panels are parts of one gel displayed at different exposures. (d) Sixteen hours post-transfection, percentages of 293T cells expressing BPLF1, BXLF1, BSLF1 or the N-terminal fragment of BPLF1 were determined by intracellular FACS staining for the Flag-tag. (e) Immunoblot of part of the gel in c probed with an anti-Flag Ab to detect transfected BXLF1 and BSLF1 (sequential staining following 1F2, see in f). (f) Immunoblot of the gel in c probed with the BPLF1-specific mouse monoclonal Ab 1F2. Both panels are part of one gel presented at different exposures.

293T cells were transiently transfected with a firefly luciferase reporter construct responsive to either NF-κB activation or IFNβ promoter activity, an HSV TK-promoter driven Renilla luciferase plasmid (for normalization), and plasmids encoding HA-BPLF1, Flag-BXLF1, Flag-BSLF1 or cellular control DUB A20. (a) An NF-κB responsive firefly luciferase reporter and 4 or 8 ng of vectors encoding the EBV or control proteins were cotransfected in 293T cells and the TLR signaling cascade was initiated by expressing adaptor protein MyD88. Sixteen hours post-transfection, cells were lysed and luciferase activity was determined. In addition, protein expression was analyzed in post-nuclear cell lysates by immunoblot using anti-HA and anti-Flag Abs; β-actin served as loading control. (b) 293-TLR2/CD14 cells were cotransfected with an NF-κB responsive reporter and 32 ng of plasmids encoding the indicated proteins. Starting at 16 hours post-transfection, cells were stimulated with 10 ng/mL MALP-2 or 6.5×10⁶ EBV particles/mL for 7 hours, after which they were lysed and luciferase activity was measured. (c) Similarly, 293-TLR3 cells were cotransfected with 16 ng of the indicated genes together with an NF-κB responsive reporter (left panel) or a reporter under control of the IFNβ promoter (right panel). Starting 16 hours post-transfection, cells were stimulated for 7 hours with 10 µg/mL poly(I∶C), after which they were lysed and luciferase activity was determined. Data are presented as percentages firefly luciferase activity relative to stimulated control samples and normalized for transfection efficiency using Renilla luciferase values, mean ± SD. (d) 293-TLR2/CD14 cells were transfected with empty control vector or plasmids encoding EBV BPLF1 or cellular A20. Starting at 24 hours post-transfection, cells were stimulated with 10 µg/mL MALP2 or 6.5×10⁶ EBV particles/mL for 8 hours. IL-8 secretion in the culture supernatants was determined by ELISA.

293T cells were transiently transfected with an empty vector control or plasmids encoding Flag-tagged BPLF1 or mutants thereof (BPLF1_C61A, BPLF1_R86/R90, or BPLF1_G86/G90). (a) Sixteen hours post-transfection, the percentage of cells expressing BPLF1 was determined by intracellular flow cytometric analysis using an anti-Flag Ab. (b, upper panel) Post-nuclear cell lysates were prepared and immunoblotted using an anti-Flag Ab. The asterisk marks a smaller active fragment observed upon expression of BPLF1. (b, middle panel) Active DUBs in these cell lysates were labeled using the fluorescent Ub-VME probe (causing a concomitant size increase of 10 kDa) and visualized by in-gel imaging. (b, lower panel) Immunoblot of the gel in c probed with BPLF1-specific Ab 2E5. Unlabeled BPLF1 migrates at 36 kDa and 32 kDa, while the upper two bands of 46 kDa and 42 kDa represent probe-bound BPLF1. (c) 293T cells were cotransfected with HA-ubiquitin and BPLF1 variants. After 40 hours, total lysates were prepared and immunoblotted for HA-Ub-protein adducts using an anti-HA Ab (left panel; right panel shows the quantification of average staining intensities from four independent experiments). Expression levels of BPLF1 variants were assessed using an anti-Flag Ab; β-actin served as loading control. (d) 293T cells were cotransfected with vectors encoding TLR2, an NF-κB responsive firefly luciferase reporter, HSV TK-driven Renilla luciferase for normalization, and BPLF1 variants (16 ng). Following treatment with 10 ng/mL MALP-2 for 7 hours, cells were lysed and luciferase activity was measured. The data are presented as percentage firefly luciferase activity relative to stimulated control sample and normalized for transfection efficiency using Renilla luciferase values, mean ± SD.

(a) 293-TLR2/CD14 cells and control 293-CD14 cells were transiently transfected with plasmids encoding Flag-tagged BPLF1, catalytically inactive mutant BPLF1_C61A, or cellular control DUB A20. At 40 hours post-transfection, cells were treated with 10 ng/mL MALP-2 for indicated time periods and IκBα levels in post-nuclear cell extracts were determined by immunoblotting with an anti-IκBα Ab. Expression of transfected Flag-tagged proteins was assessed by immunoblotting with an anti-Flag Ab; TfR served as loading control. (b) Schematic representation of the signaling cascade that induces NF-κB following TLR activation. Ub in green is K63-linked, Ub in purple is K48-linked. (c) 293T cells were cotransfected with an NF-κB responsive firefly luciferase reporter, HSV TK promoter-driven Renilla luciferase for normalization, and plasmids expressing BPLF1, BPLF_C61A, or controls IκBα and A20 (13.5 ng). TLR signaling was induced by expression of activator proteins MyD88, IRAK1, TRAF6, or IKKα. After 16 hours of transfection, luciferase activity was measured. Results are depicted as percentage firefly luciferase activity normalized using Renilla luciferase values relative to stimulated control sample, mean ± SD.

(a) Fluorescence micrographs of 293T cells that were cotransfected with plasmids encoding Flag-TRAF6 and HA-BPLF1 (upper panel), NEMO and Flag-BPLF1 (middle panel), or IκBα and Flag-BPLF1 (lower panel). Sixteen hours post-transfection, signaling intermediates (column 2) and BPLF1 (column 3) were labeled using anti-Flag (red; for TRAF6 in upper panel; for BPLF1 in middle and lower panels), anti-BPLF1 (green), anti-NEMO (green), and anti-IκBα (green) Abs as indicated. Nuclei were visualized using TO-PRO-3 (blue, column 1). Column 4 and 5 show merged pictures from red and green channels without and with nuclear stain (blue), respectively. Scale bar 50 µm. (b) 293T cells were cotransfected with plasmids encoding either Flag-TRAF6 (upper panel) or His-NEMO (lower panel) together with HA-ubiquitin and BPLF1, BPLF1_C61A, or A20. Expression of Flag-tagged proteins was assessed by immunoblotting (IB) with an anti-Flag Ab. TRAF6 and NEMO were precipitated (IP) from post-nuclear lysates and detected using an anti-TRAF6 or anti-His Ab, respectively. HA-tagged ubiquitin adducts were visualized on immunoblot using anti-HA Abs. (c) 293T cells were cotransfected with plasmids encoding HA-ubiquitin together with Flag-TRAF6 (left panel), His-NEMO (middle panel), or IκBα (right panel). IκBα-transfected cells were additionally treated with TNFα and the proteasome inhibitor MG132 for 2 hours before lysis. Twenty-four hours post-transfection, TRAF6, NEMO, and IκBα were precipitated from post-nuclear cell lysates and incubated in vitro with separately purified Flag-BPLF1, Flag-BPLF1_C61A, or Flag-A20 proteins for 4 hours at 37°C. Flag-tagged DUBs were detected by immunoblotting with anti-Flag Ab and signaling intermediates were visualized using anti-Flag (TRAF6), anti-His (NEMO), or anti-IκBα Abs. HA-ubiquitin adducts were detected using an anti-HA Ab.

EBV⁺ AKBM cells were treated with anti-human IgG (αIgG) to induce productive infection. (a) At the indicated times post-induction, percentages of AKBM cells undergoing productive infection were determined by flow cytometric analysis of ratCD2-GFP reporter expression. (b) EBV protein expression in post-nuclear cell lysates was determined by immunoblotting with Abs specific for BZLF1 (immediate early, IE), BGLF5 (early, E), and gp42 (late, L). β-actin served as loading control. (c) Immunoblot of post-nuclear lysates of EBV B95.8 particles (lane 1), EBV⁻ AK31 cells (lanes 2–4), and EBV⁺ AKBM cells (lanes 5–7). EBV⁺ AKBM cells and EBV⁻ control AK31 cells were treated with αIgG Abs for 24 hours, resulting in productive infection in 26% of AKBM cells; AK31 cells expressed ratCD2-GFP constitutively in ∼40% of the population (data not shown). Phosphonoacetic acid (PAA, 300 µg/mL) treatment starting 1 hour prior to anti-IgG treatment was used to inhibit late protein expression; β-actin served as loading control.

During EBV infection, EBV components are sensed by host TLRs (a). These receptors activate signaling pathways through adaptor molecules MyD88 and TRIF, culminating in activation of transcription factor NF-κB (b). NF-κB induces production of proinflammatory cytokines (c) and obstructs viral replication. TLR signaling pathways are extensively regulated by ubiquitination, a process governed by cellular ubiquitin ligases and deubiquitinases (e.g. A20, d). The EBV-encoded DUB BPLF1 counteracts TLR-mediated NF-κB activation and can interfere with K63- and K48-linked ubiquitination of signaling intermediates, for example on TRAF6, NEMO, and IκBα (e). BPLF1 is expressed as a full-length protein during the late phase of productive EBV infection, is incorporated into the tegument of viral particles (f), and can subsequently be released into newly infected cells (g). It is as yet unclear where BPLF1 is processed to yield a shorter active fragment of ca. 280 aa (see ). Thus, (processed) BPLF1 could exert its immunomodulatory functions towards TLR signaling not only in EBV-producing B cells (h), but also during primary infection (g).