Inhibition of S-phase cyclin-dependent kinase activity blocks expression of Epstein-Barr virus immediate-early and early genes, preventing viral lytic replication

Abstract

The induction of lytic replication of the Epstein-Barr virus (EBV) completely arrests cell cycle progression, in spite of elevation of S-phase cyclin-dependent kinase (CDK) activity, thereby causing accumulation of hyperphosphorylated forms of retinoblastoma (Rb) protein (A. Kudoh, M. Fujita, T. Kiyono, K. Kuzushima, Y. Sugaya, S. Izuta, Y. Nishiyama, and T. Tsurumi, J. Virol. 77:851-861, 2003). Thus, the EBV lytic program appears to promote specific cell cycle-associated activity involved in the progression from G1 to S phase. We have proposed that this provides a cellular environment that is advantageous for EBV productive infection. Purvalanol A and roscovitine, inhibitors of S-phase CDKs, blocked the viral lytic replication when cells were treated at the early stage of lytic infection, while well-characterized inhibitors of enzymes, such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C, known to be involved in BZLF1 gene expression did not. Inhibition of CDK activity resulted in the accumulation of the hypophosphorylated form of Rb protein and inhibition of expression of EBV immediate-early and early proteins. Cycloheximide block-and-release experiments clearly demonstrated that even in the presence of enough amounts of the BZLF1 protein, purvalanol A blocked expression of lytic viral proteins at transcription level. Furthermore, reporter gene experiments confirmed that BZLF1-induced activation of early EBV promoters was impaired in the presence of the CDK inhibitor. We conclude here that the EBV lytic program promotes specific cell cycle-associated activity involved in the progression from G1 to S phase because the S-phase-like cellular environment is essential for the expression of immediate-early and early genes supplying the viral replication proteins and hence for lytic viral replication.

FIG. 1.

(A) Specific inhibition of EBV lytic replication by the CDK inhibitors roscovitine and purvalanol A. Tet-BZLF1/B95-8 cells were cultured in the presence of 1 μg of doxycycline/ml with drug-free medium (control) or media containing each indicated drug and harvested at the indicated times. The drug concentrations used in the experiments were as follows: 10 μM bisindolylmaleimide I, 15 μM LY-294002, 20 μM mevinolin, 50 μM roscovitine, and 15 μM purvalanol A. To determine viral DNA synthesis, slot blot assays for viral DNA were performed as described in Materials and Methods. Signal intensity was quantified with an Image guider (Fujifilm), and the copy numbers of viral genome per cell were calculated. (B) Effect of bisindolylmaleimide I, LY-294002, mevinolin, and purvalanol A on the proliferation of Tet-BZLF1/B95-8 cells. Tet-BZLF1/B95-8 cells were seeded in flasks (10⁷ cells/flask) with drug-free medium (control) or media containing each indicated drug. The numbers of cells were counted with a hemocytometer at the indicated times, and the data were plotted in the graph. The drug concentrations used in the experiments were the same as those described for panel A.

FIG. 2.

Inhibition of EBV lytic replication by purvalanol A is reversible. Tet-BZLF1/B95-8 cells were cultured with doxycycline (1 μg/ml) in the presence (○, •) or absence (♦) of purvalanol A (15 μM). Aliquots of cells treated with purvalanol A were also transferred to purvalanol A-free fresh medium at 24 h postinduction (dashed line). This medium change is indicated by the arrow. Cells were harvested at the indicated times, and viral DNA synthesis was determined by slot blot assay as described in Materials and Methods.

FIG. 3.

(A) Dose dependence of purvalanol A effects on the proliferation of Tet-BZLF1/B95-8 cells. Tet-BZLF1/B95-8 cells were seeded in 35-mm-diameter petri dishes (1.0 × 10⁶ cells/dish). The cells were incubated for 48 h in RPMI 1640 medium containing the indicated concentrations of purvalanol A. The cells were harvested, and total cell numbers were counted with a hemocytometer. (B) Effects of purvalanol A on the phosphorylation state of Rb protein. Cells were cultured in the presence of indicated concentrations of purvalanol A and harvested after 48 h. Clarified cell lysates were prepared, separated by SDS-7.5% PAGE, and applied for Western blot analyses with anti-Rb protein polyclonal antibody. pRB and ppRB are slower-migrating hyperphosphorylated forms of Rb protein. The faster-migrating band is the hypophosphorylated form of Rb protein and is designated as RB.

FIG. 4.

The CDK inhibitors purvalanol A and roscovitine inhibit EBV lytic replication when added at least until 9 h postinduction. Tet-BZLF1/B95-8 cells were treated with 1 μg of doxycycline/ml for 72 h. Purvalanol A (15 μM) (A) or roscovitine (50 μM) (B) was added at the indicated times (0, 3, 6, 9, 24, and 48 h) postinduction, and the cells were harvested at 72 h postinduction and viral DNA synthesis was determined by slot blot assay as described in Materials and Methods (dashed lines, ○). Signal intensity was quantified with an Image guider (Fujifilm). An additional series of Tet-BZLF1/B95-8 cells with productive infection was incubated continuously in CDK inhibitor-free medium and harvested at the indicated times (control; •).

FIG. 5.

Effects of purvalanol A on the expression of lytic viral proteins and phosphorylation of Rb protein during EBV lytic infection. (A) Tet-BZLF1/B95-8 cells were treated with (+) or without (−) 5 μg of doxycycline/ml for 48 h and purvalanol A (20 μM) was added to the culture immediately thereafter (0 h). The cells were harvested at the indicated times, and clarified cell lysates were prepared. Phosphorylation states of the Rb protein were analyzed by Western blotting with anti-Rb protein monoclonal antibody. pRB and ppRB are slower-migrating hyperphosphorylated forms of Rb protein. The faster-migrating band is the hypophosphorylated form of Rb protein and is designated as RB. (B) Purvalanol A (20 μM) was added to the culture of doxycycline-treated Tet-BZLF1/B95-8 cells at 0, 3, 6, 9, and 24 h postinduction, and the cells were harvested at the indicated times. Clarified cell lysates were separated by SDS-PAGE and applied for Western blot analyses with each antibody as indicated on the right. The proteins were detected with an enhanced chemiluminescence detection system (Amersham). Images were processed by LumiVision PRO (Aisin/Taitec, Inc.) with a cooled CCD camera and assembled using an Apple G4 computer with Adobe Photoshop 5.0. Signal intensity was quantified with a LumiVision image analyzer as described in Materials and Methods. IE, immediate-early proteins; E, early proteins. (C) Tet-BZLF1/AKATA cells were treated with (+) or without (−) 3 μg of doxycycline/ml and cultured for 48 h in the absence or presence of purvalanol A (5 μM). Lane 1 shows the sample prepared when the cells were treated with doxycycline at time zero. The cells were harvested, and clarified cell lysates were prepared and applied for Western blot analyses with each antibody as indicated on the right.

FIG. 6.

Effects of purvalanol A on expression of EBV immediate-early and early proteins after release from a cycloheximide block. (A) Illustration of the experimental protocol. Tet-BZLF1/B95-8 cells were treated with doxycycline (1 μg/ml). Three hours later, 50 μg of cycloheximide (CHX)/ml was added to the culture. The cells were incubated for 9 h and then removed from the CHX block by replacing the medium with drug-free medium and culturing for a further 2 h. Subsequently, the medium was replaced with one containing no drug (CHX-None), 1 μg of doxycycline/ml (CHX-Dox), 1 μg of doxycycline/ml plus 15 μM purvalanol A (CHX-Dox+Purv), or 1 μg of doxycycline/ml plus 50 μg of cycloheximide/ml (CHX-Dox+CHX), and the cells were cultured for 48 h and harvested (lanes 2, 3, 4, and 5 of panel B, respectively; lane 1 of panel B represents the sample prepared when the cells were treated with doxycycline at time zero). (B) Clarified lysates were prepared and applied for Western blot analyses with each specific antibody as indicated on the left. The proteins were detected by an enhanced chemiluminescence detection system (Amersham). Images were processed by LumiVision PRO (Aisin/Taitec, Inc.) with a cooled CCD camera and assembled using an Apple G4 computer with Adobe Photoshop 5.0. Signal intensity was quantified with a LumiVision image analyzer as described in Materials and Methods. IE, immediate-early proteins; E, early proteins.

FIG. 7.

Effects of purvalanol A on transcription of EBV immediate-early and early genes after release from a cycloheximide block. (A) Illustration of the experimental protocol. Tet-BZLF1/B95-8 cells were treated with doxycycline (1 μg/ml). At 3 h thereafter, 50 μg of cycloheximide (CHX)/ml was added to the culture. The cells were incubated for 9 h and then removed from the CHX block by replacing the medium with drug-free medium and culturing for a further 2 h. Subsequently, the medium was replaced with fresh medium containing 1 μg of doxycycline/ml and cultured for 48 h in the absence or presence of 15 μM purvalanol A [(2) and (3), respectively] and then harvested. As a control, cells were harvested before 48 h cultivation [(1)]. (B) Total RNA was extracted and applied for Northern blot analyses as described in Materials and Methods. (C) Levels of BZLF1-, BRLF1-, BALF5-, BMRF1-, and 36B4-specific transcripts (panels a to e, respectively, in Fig. 7C) were measured by Northern hybridization with ³²P-labeled probes obtained by PCR amplification of each viral and cellular DNA. Signal intensity was quantitated with an Image guider (BAS2500; Fujifilm). The levels of the specific viral and cellular gene transcripts relative to the pretreatment values were calculated and plotted in the graphs.

FIG. 8.

Purvalanol A prevents BZLF1-induced transcriptional activation of the early EBV promoters pBMRF1 and pBHRF1. Tet-BZLF1/HeLa cells were transfected with 2 μg of pBMRF1-CAT or pBHRF1-CAT and media were replaced with fresh media containing or free of purvalanol A (20 μM) at 16 h posttransfection. Twenty hours posttransfection, doxycycline was added to a final concentration of 5 μg/ml or not. Forty-eight hours after addition of doxycycline, cells were harvested and some of them were used for a CAT enzyme assay. (A) CAT activity was assayed by using the Promega CAT enzyme assay system. The total amount of n-butyryl [¹⁴C]chloramphenicol (in picomoles) was measured and plotted on the graph. Data represent averages from three independent experiments. (B) The rest of the cells were treated with lysis buffer, and clarified cell lysates were prepared. Clarified cell lysates were separated by SDS-10% PAGE and applied for Western blot analyses with the BZLF1 protein-specific antibody. The proteins were detected by an enhanced chemiluminescence detection system (Amersham). The slower-migrating band is a phosphorylated form of the BZLF1 protein. The faster-migrating band is the hypophosphorylated form.