FIG 4.

Loss of BDLF4 is responsible for the inhibition of late gene expression (A) HEK293 cells latently infected with EBV were transfected with the indicated expression vector and harvested after 96 h. After freeze-thawing and centrifugation, the supernatants were cultured with Akata(-) cells. FACS analysis was performed to count GFP-positive cells. (B) Levels of viral proteins. HEK293 cells with recombinant EBV were transfected with the indicated vector. Whole-cell lysates were prepared at 0 and 72 h and analyzed by Western blotting with anti-BALF2, anti-BDLF4, anti-BZLF1, anti-BALF4 (gB), and anti-tubulin. (C) Levels of viral late mRNAs. HEK293 cells with recombinant EBVs were transfected with the indicated vector. The mRNAs were prepared at 72 h and analyzed by real-time RT-PCR. (D) Association of BDLF4 with other late gene regulators. HEK293 cells were transfected with expression vectors for Flag-BDLF4, Myc-BGLF3, hemagglutinin-BcRF1 (HA-BcRF1), HA-BVLF1, and HA-BDLF3.5. Immunoprecipitation (IP) was carried out using anti-Flag antibody, and the results were detected by Western blotting using anti-Myc, anti-HA, and anti-Flag antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a negative control. The arrowhead indicates immunoglobulin.