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. 2019 Apr 3;93(8):e01707-18. doi: 10.1128/JVI.01707-18

FIG 4.

FIG 4

CDK inhibitors regulate the expression of late genes during EBV lytic replication. (A) qPCR of viral RNAs (center) and viral replication levels (right) in HEK293/EBV(WT) cells treated with CDK inhibitors at 48 h after the induction of lytic replication. Cells were transfected with the BZLF1 expression plasmid and were treated with CDK 2/9 inhibitor (CDK2/9i; 500 nM) or alsterpaullone, 2-cyanoethyl (A2CE; 1 μM) as indicated on the schedule (left). Results shown are means ± SDs from three independent experiments. Double asterisks, P < 0.01; n.s., not significant. (B) qPCR analyses of viral RNA levels (center) and viral replication levels (right) in HEK293/EBV(WT) cells treated with CDK inhibitors 48 h after the induction of lytic replication. Cells were transfected with a BZLF1-expressing plasmid and were treated with CDK2/9i (500 nM) or A2CE (1 μM) as indicated on the schedule (left). Results shown are means ± SDs from three independent experiments. Double asterisks, P < 0.01. (C and D) HEK293/EBV(WT) cells were transfected with a TATT-oriLyt-Luc reporter plasmid together with pSV40-Rluc as an internal control and a BZLF1 expression plasmid as indicated. DMSO or a CDK inhibitor was added to the medium at 24 h p.t. (C) Lysates harvested from the cells at 48 h p.t. were subjected to immunoblot analysis with the indicated antibodies. BZLF1 is an IE gene; BMRF1 and BALF2 are E genes; and BRRF2, BALF4 (gB), and BKRF4 are L genes (11, 48, 55, 56). (D) Lysates were also analyzed using luciferase reporter assays. The results are presented as means ± SDs from three independent transfections after normalization to the internal control (Renilla luciferase activity). Double asterisks, P < 0.01. (E) HEK293/EBV(WT) cells were first transfected with BZLF1 expression vectors and then treated with CDK inhibitors. The virus yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments and are relative to the virus yield of BZLF1 with DMSO treatment (infectivity value, 1). Double asterisks, P < 0.01.