FIG. 4.

BGLF4 interacts with EBNA1. (A) GST affinity assay. GST-BGLF4-bound glutathione beads were incubated with purified, bacterially expressed V5-EBNA1c in kinase buffer with or without ATP or γ-S-ATP. The control reaction lacked GST-BGLF4. Bound V5-EBNA1c was detected by Western blotting using anti-V5 antibody. The blots were probed with anti-GST (α-GST) antibody to show loading of GST-BGLF4. (B) Relocation of V5-EBNA1c into the nucleus by BGLF4. Immunofluorescence assays in transfected HeLa cells are shown. EBNA1c lacks a nuclear localization signal and has a cytoplasmic localization (RHOD) (top). In the presence of HA-BGLF4 or HA-mtBGLF4 (FITC) (middle and bottom), EBNA1c was relocated into the nucleus (RHOD and Merge) (middle and bottom). Nuclei were visualized with DAPI. (C) Coprecipitation of GFP-EBNA1 with HA-BGLF4 and HA-mtBGLF4. HA-BGLF4 proteins present in the direct α-HA and indirect α-EBNA1 immunoprecipitates generated from extracts of cotransfected HeLa cells were detected by probing a Western blot (WB) with α-HA antibody. IP, immunoprecipitation. (D) Coprecipitation of endogenous EBNA1 with BGLF4 using extracts from a doxycycline (Dox)-inducible BGLF4-expressing Akata cell line. (Left) Western blots showing expression of EBNA1 and BGLF4 before and after treatment with doxycycline. (Right) Western blot showing BGLF4 protein present in the direct anti-BGLF4 immunoprecipate, the cell extract, and the indirect anti-EBNA1 immunoprecipitate. BGLF4 was detected with anti-BGLF4 antibody. (E) Western blots showing coprecipitation of BGLF4 with EBNA1 in lytically induced Akata cells.