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. 2023 Feb 21;19(2):e1011186. doi: 10.1371/journal.ppat.1011186

Fig 10. BPLF1 suppresses IFN-β production in EBV-infected cells.

Fig 10

(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.