Fig 1.
(A) Functional screening for IFN-β antagonists. HEK293 cells were transfected with a Firefly luciferase reporter plasmid driven by IFN-β-promoter (IFNB-Luc), a control Renilla luciferase reporter plasmid driven by thymidine kinase promoter (TK-Luc) serving to normalize for transfection efficiency, the plasmids for EBV protein expression library, together with cGAS and STING expression plasmids. Cells were harvested 24 hours post-transfection for dual-luciferase assay. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. (B-D) BPLF1 inhibits cGAS-STING-, RIG-I- and TBK1-induced IFNβ-promoter activation. HEK293 cells were transfected with IFNB-Luc, TK-Luc, and increasing doses of BPLF1 expression plasmid. IFNB-Luc reporter expression was stimulated with expression plasmids for cGAS + STING (B), RIG-I N (C) and TBK1 (D). Cells were harvested 24 hours post-transfection for dual-luciferase assay. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance for the difference between the indicated groups was analyzed using two-tailed Student’s t-test for paired samples and the ranges of the p values were indicated (*: p < 0.05; **: p < 0.01; and ***: p < 0.001).
Fig 2.
(A-D) Specific inhibition of cGAS-STING activity by BPLF1. HEK293 cells were transfected with IFNB-Luc, TK-Luc, either one or two doses of BPLF1 expression plasmid, and expression plasmids for cGAS + STING (A), IRF3 (B) or IRF3-5D (C). Alternatively, a Firefly luciferase reporter driven by the SV40-promoter (SV40-Luc) and TK-Luc promoter were co-transfected with BPLF1 expression plasmid into HEK293 cells (D). Cells were harvested 24 hours post-transfection for dual-luciferase assay. (E) A schematic diagram depicting the targeting regions of the two gRNAs (guide-1 and guide-2) on the EBV genome was presented together with dCas9, four tandem copies of HSV-1 VP16 molecules (VP64) and the activator helper complex. (F) The cDNA sequences of the two gRNAs. (G) Induction of BPLF1 expression by CRISPR-a. HEK293M81 cells were transfected with lentiMPHv2 and lentiSAMv2 carrying guide-1 and guide-2. The total cellular RNA was harvested 48 hours post-transfection and the BPLF1 mRNA was measured by RT-qPCR. The BPLF1 mRNA expression levels were normalized to those of endogenous GAPDH transcript. (H) IFN antagonism of BPLF1 induced to express from EBV genome by CRISPR-a. HEK293M81 cells were transfected with IFNB-Luc, TK-Luc, guide-1 or guide-2 plasmids and expression plasmids for cGAS + STING, RIG-I N and TBK1. (I) Dose-dependent IFN antagonism of BPLF1 expressed from EBV genome. HEK293M81 cells were transfected with IFNB-Luc, TK-Luc, increasing doses of guide-2 plasmid, and TBK1 expression plasmid for dual luciferase assays. (J-L) BPLF1 inhibits cGAS-STING-, RIG-I- and TBK1-induced activation of IRF3-binding elements. HEK293 cells were transfected with a Firefly luciferase reporter plasmid driven by IRF3-Luc, TK-Luc, increasing doses of BPLF1 expression plasmid, and expression plasmids for cGAS + STING (J), RIG-I N (K) or TBK1 (L). IκB super-repressor was also over-expressed in (J). Cells were harvested 24 hours post-transfection for dual-luciferase assay. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance among selected samples was analyzed using two-tailed Student’s t-test for paired samples and the ranges of the p values were indicated (*: p < 0.05; **: p < 0.01; and n.s.: not significant or p > 0.05).
Fig 3.
Requirement of deubiquitinase domain (DUB) for IFN-inhibitory effect of BPLF1.
(A) Expression of BPLF1 C61A mutant. BPLF1 WT and C61A were transiently expressed in HEK293 cells. Endogenous α-tubulin was used for normalization. (B-D) IRF3 activation by C61A mutant. The indicated combinations of BPLF1 expression plasmid, mutant C61A expression construct as well as IRF3-Luc and TK-Luc reporter plasmids were transfected into HEK293 cells together with cGAS + STING (B), RIG-I N (C) or TBK1 (D) expression plasmids. Cells were harvested 24 hours post-transfection for dual-luciferase assay. (E) Effect of C61A on IFN-β induction. HEK293 cells were transfected with cGAS, STING, BPLF1 and mutant C61A expression plasmids. The total cellular RNA was harvested 48 hours post-transfection and the IFN-β transcripts expression levels were measured by RT-qPCR. The IFNβ transcript expression levels were normalized to those of endogenous GAPDH transcript. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. (F, G) The DUB activity of BPLF1 promotes EBV infection. BPLF1 and C61A expression plasmids were transfected into HEK293 cells together with cGAS + STING expression plasmids (panels 1–3) and TBK1 expression plasmid (panels 4–6). After 24 hours post- transfection, cells were infected with 1 m.o.i. of freshly prepared EBV M81. Microscopic images were captured (F) and GFP signals were analyzed by flow cytometry 48 hours post-infection (G). The percentages of GFP+ cells were normalized to the mock infection group. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance among selected samples was analyzed using two-tailed Student’s t-test for paired samples and the ranges of the p values were indicated (*: p < 0.05; and **: p < 0.01; ***: p < 0.001).
Fig 4.
BPLF1 perturbs STING ubiquitination.
(A) Influence of BPLF1 and C61A on STING ubiquitination. BPLF1, C61A, cGAS and STING expression plasmids were transfected into HEK293 cells together with HA-Ub-WT and HA-Ub-K0. (B) Influence of BPLF1 and C61A on STING ubiquitination of different types. BPLF1, C61A, cGAS and STING expression plasmids were transfected into HEK293T cells together with HA-Ub-WT and HA-K63-Ub, HA-K48-Ub and HA-K27-Ub. (C) Influence of BPLF1 and C61A on K48R- and K63R-linked ubiquitination of STING. BPLF1, C61A, cGAS and STING expression plasmids were transfected into HEK293T cells together with HA-Ub-WT, HA-K63R-Ub and HA-K48R-Ub. Cells were harvested 24 hours after transfection for co-immunoprecipitation. The Flag-tagged STING molecules were pulled down by anti-Flag antibodies. The bound fraction of the immunoprecipitates (IP) and the total lysates (input) were analyzed by Western blotting (WB) with anti-HA, anti-Flag and anti-β-tubulin antibodies. Long exposure was conducted to visualize proteins in the upper panel and short exposure was conducted to visualize other proteins in the lower panel.
Fig 5.
BPLF1 perturbs TBK1 ubiquitination.
(A) Influence of BPLF1 and C61A on TBK1 ubiquitination. BPLF1, C61A and TBK1 expression plasmids were transfected into HEK293T cells together with HA-Ub-WT and lysine-free ubiquitin (HA-Ub-K0). (B) Influence of BPLF1 and C61A on TBK1 ubiquitination of different types. BPLF1, C61A and TBK1 expression plasmids were transfected into HEK293T cells together with HA-Ub-WT and HA-K63-Ub and HA-K48-Ub. Cells were harvested 24 hours after transfection for co-immunoprecipitation. The Flag-tagged TBK1 molecules were pulled down by anti-Flag antibodies. The bound fraction of the immunoprecipitates (IP) and the total lysates (input) were analyzed by Western blotting (WB) with anti-HA, anti- Flag and anti-β-tubulin antibodies. Long exposure was conducted for visualizing proteins in the upper panel and short exposure was conducted for visualizing proteins in the lower panel.
Fig 6.
BPLF1 binds to STING, but not TBK1.
(A, B) Co-immunoprecipitation of BPLF1 and STING. Myc-BPLF1 and Flag-STING expression plasmids were expressed in HEK293T cells. (C) No association between BPLF1 and TBK1. Myc-BPLF1 and Flag-TBK1 were expressed in HEK293T cells. Cells were harvested 24 hours after transfection for co-immunoprecipitation. The Flag-tagged STING and TBK1 proteins were pulled down by anti-Flag antibodies. The bound fraction of the immunoprecipitates (IP) and the total lysates (input) were analyzed by Western blotting (WB) with anti-Myc, anti-Flag and anti-α-tubulin antibodies.
Fig 7.
BPLF1 mitigates TBK1-induced dimerization of IRF3.
IRF3 dimerization assay by native PAGE. BPLF1 and mutant C61A were transiently overexpressed in HEK293 cells together with TBK1 and V5-IRF3. Cells were harvested 48 hours after transfection. The cell lysates were subjected to SDS-PAGE and native PAGE. The lysates were analyzed by Western blotting by anti-V5, anti-Flag, anti-TBK1 and anti-α-tubulin antibodies.
Fig 8.
Prevalence of BPLF1 in different EBV+ cells and the effect of BPLF1 activation on IFN-β level in gastric cancer cells.
(A-E) NP460EBV, HEK293p2089, HEK293M81, NPC43 and AGS-BX1 cells were seeded into 6-well plates. Cells were treated with either 40 ng/ml TPA (T) and 1.5 nM sodium butyrate (S) or 40 ng/ml TPA. The total cellular RNA was harvested 48 hours post-transfection and the BPLF1 (A), LMP1 (B), Zta (D) and Rta (E) transcript expression levels were determined by RT-qPCR. The relative expression levels were normalized to endogenous GAPDH transcript expression levels. (C) The relative expression levels of BPLF1 transcript were further normalized with the relative expression levels of LMP1 transcript. (F) AGS-BX1 cells were transfected with cGAS and STING expression plasmids, lentiSAMv2 carrying guide-1 and guide-2, and lentiMPHv2. Total cellular RNA was harvested 48 hours post-transfection and the IFN-β transcript expression levels were measured by RT-qPCR. The IFN-β transcript expression levels were normalized to the endogenous GAPDH transcript level. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance among selected samples were analyzed using two-tailed Student’s t-test for paired samples and the range of p values was indicated (*: p < 0.05).
Fig 9.
Characterization of recombinant EBV with a catalytically dead BPLF1.
(A) Schematic diagram of the flanking primers designed for PCR genotyping of the fragments around position 61 at the N-terminus of BPLF1 in EBV BAC. (B) PCR amplification of the fragments of unedited SW105-p2089 clone, galK inserted clone and C61A mutant clone. (C) Sequencing results of the unedited SW105-p2089 clone and C61A mutant virus clone. (D) BamHI restriction patterning of mutant C61A cloned 1 and 2 as well as unedited EBV BAC SW105-p2089. 10 μg and 12 μg of the EBV BACs were digested and the BamHI restriction patterning was evaluated. Nanopore sequencing of the BACs has been conducted and no undesirable change was spotted in the mutant virus.
Fig 10.
BPLF1 suppresses IFN-β production in EBV-infected cells.
(A) BPLF1 mRNA expression in EBV+ cells. HEK293, NP460EBV, HEK293p2089#1–2, HEK293p2089-C61A#1–4 cells were seeded into 6-well plates. The total cellular RNA was harvest 48 hours after transfection and the BPLF1 transcript expression levels were determined by RT-qPCR and the relative BPLF1 transcript expression levels were normalized to endogenous GAPDH transcript levels. (B) STING ubiquitination in cells carrying BPLF1-inactive EBV. HEK293, HEK293p2089 and HEK293p2089-C61A#1–4 cells were seeded into 60mm dishes. Cells were harvested 48 hours after transfection for co-immunoprecipitation. The Flag-tagged STING molecules were pulled down by the anti-Flag antibodies. The bound fraction of the immunoprecipitates (IP) and the total lysates (input) were analyzed by Western blotting (WB) with anti-HA, anti-Flag and anti-β-tubulin antibodies. (C) IFNB-Luc activation in cells carrying BPLF1-inactive EBV. IFNB-Luc and TK-Luc reporter plasmids were transfected into HEK293, HEK293p2089#1–2, HEK293p2089-C61A#1–4 cells together with cGAS and STING expression plasmids. (D) IFN-β induction in cells carrying BPLF1-inactive EBV. Cells were harvested 24 hours post-transfection for dual-luciferase assay. HEK293, HEK293p2089#1–2, HEK293p2089-C61A#1–4 cells were transfected with cGAS and STING expression plasmids. The total cellular RNA was harvested 48 hours post-transfection and the IFNβ transcripts expression levels were measured by RT-qPCR. The IFN-β transcript expression levels were normalized by the endogenous GAPDH transcripts level. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance among selected samples were analyzed using two-tailed Student’s t-test for paired samples and the p- values were indicated. (E) IFNB-Luc activation in cells stably carrying BPLF1-inactive EBV. WT p2089 and BPLF1C61A p2089 EBV BAC were transfected into HEK293 cells for stable cell construction. The transfected cells were selected with hygromycin for about a week and the survival cells were then subject for IFN-β analysis. The stable cells were transfected with TBK1 or cGAS and STING for IFN-β production. TPA is added at 24 hours post-transfection to stimulate the lytic transcript production. Cells were harvested in the next day and RT-qPCR were performed to measure the IFN-β transcript level. GAPDH was used for normalization.