De novo protein synthesis is required for lytic cycle reactivation of Epstein-Barr virus, but not Kaposi's sarcoma-associated herpesvirus, in response to histone deacetylase inhibitors and protein kinase C agonists

Abstract

The oncogenic human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are latent in cultured lymphoma cells. We asked whether reactivation from latency of either virus requires de novo protein synthesis. Using Northern blotting and quantitative reverse transcriptase PCR, we measured the kinetics of expression of the lytic cycle activator genes and determined whether abundance of mRNAs encoding these genes from either virus was reduced by treatment with cycloheximide (CHX), an inhibitor of protein synthesis. CHX blocked expression of mRNAs of EBV BZLF1 and BRLF1, the two EBV lytic cycle activator genes, when HH514-16 Burkitt lymphoma cells were treated with histone deacetylase (HDAC) inhibitors, sodium butyrate or trichostatin A, or a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine. CHX also inhibited EBV lytic cycle activation in B95-8 marmoset lymphoblastoid cells by phorbol ester phorbol-12-myristate-13-acetate (TPA). EBV lytic cycle induction became resistant to CHX between 4 and 6 h after application of the inducing stimulus. KSHV lytic cycle activation, as assessed by ORF50 mRNA expression, was rapidly induced by the HDAC inhibitors, sodium butyrate and trichostatin A, in HH-B2 primary effusion lymphoma cells. In HH-B2 cells, CHX did not inhibit, but enhanced, expression of the KSHV lytic cycle activator gene, ORF50. In BC-1, a primary effusion lymphoma cell line that is dually infected with EBV and KSHV, CHX blocked EBV BRLF1 lytic gene expression induced by TPA and sodium butyrate; KSHV ORF50 mRNA induced simultaneously in the same cells by the same inducing stimuli was resistant to CHX. The experiments show, for the cell lines and inducing agents studied, that the EBV BZLF1 and BRLF1 genes do not behave with "immediate-early" kinetics upon reactivation from latency. KSHV ORF50 is a true "immediate-early" gene. Our results indicate that the mechanism by which HDAC inhibitors and TPA induce lytic cycle gene expression of the two viruses differs and suggest that EBV but not KSHV requires one or more proteins to be newly synthesized between 4 and 6 h after application of an inducing stimulus.

FIG. 1.

CHX inhibits induction of EBV BRLF1 and BZLF1 mRNAs by HDAC inhibitors and a DNA methyltransferase inhibitor in HH415-16 cells. Cells were untreated (−) or treated (+) for 8 h with chemicals to induce the EBV lytic cycle, NaB (A), TSA (B), or 5Aza2′dC (AZA C) (C). CHX was added to one set of untreated or chemically treated cultures. Total cellular RNA was prepared at 0 h or 8 h. A Northern blot was probed with Z(301), which detects both BRLF1 (Rp) and BZLF1 (Zp) mRNAs. The blots were reprobed for RNase P to control for RNA loading. The relative levels of the BRLF1 3.0-kb and BZLF1 1.0-kb mRNAs were determined by densitometry and corrected for RNase P expression.

FIG. 2.

Quantitation of the inhibitory effect of CHX on EBV lytic cycle induction by real-time reverse transcriptase PCR(QRT-PCR). (A) Standard curve for QRT-PCR detection of BRLF1 mRNA. (B and C) HH514-16 cells were untreated (−) or treated (+) for various lengths of time with EBV lytic cycle inducing agents, either TSA (B) or 5Aza2′dC (AZA C) (panel C). One set of cultures exposed to the lytic cycle inducing agent for different lengths of time was also treated with CHX. RNA was harvested 9 h after the start of the experiment. The relative levels of BRLF1 mRNA were measured by QRT-PCR. S.I., stimulation index.

FIG. 3.

Determination of time after addition of the inducing agent when CHX inhibits EBV lytic cycle activation in HH514-16 cells. Cells were untreated (lanes 1 and 2) or treated (lanes 3 to 7) with NaB (A), TSA (B), or AZAC (C). CHX was added with the inducing agent or at 2-h intervals thereafter. RNA was prepared at 0 h (lane 1) or 8 h (lanes 2 to 7) after the start of the experiment. Northern blots were probed with Z(301); mRNA abundance was quantitated by densitometry. Rp, BRLF1; Zp, BZLF1.

FIG. 4.

CHX inhibits EBV early lytic cycle mRNA expression in B95-8 cells treated with phorbol ester. B95-8 cells received (+) (lanes 4 to 8) or did not receive (−) (lanes 1 to 3) TPA. CHX was added for the durations indicated (0 to 8 h). Total cellular RNA was harvested at 0 h (lane 1) or 8 h (lanes 2 to 8). Northern blots were probed with Z(301) and quantitated by densitometry. Rp, BRLF1; Zp, BZLF1.

FIG. 5.

Activation of KSHV ORF50 mRNA in HH-B2 cells by NaB is resistant to CHX. (A) Kinetics of activation ORF50 mRNA in HH-B2 cells by two HDAC inhibitors. RNA prepared at the times indicated after exposure to NaB, AZAC, or TSA was analyzed by Northern blotting with the probe KSR(243), which detects the 3.6-kb ORF50 mRNA (52). (B) HH-B2 cells were untreated (lanes 1 and 2) or treated with NaB (lanes 3 to 7). CHX was added for the durations indicated. RNA harvested at 8 h was analyzed by Northern blotting with the KSR(243) probe.

FIG. 6.

CHX enhances expression of ORF50 mRNA in HH-B2 cells treated with HDAC inhibitors. (A) Standard curve for QRT-PCR detection of ORF50 mRNA. (B and C) HH-B2 cells were untreated (−) or treated (+) with NaB (B) or TSA (C) for different durations. One set of cultures received CHX for the duration of the experiment (3 h). RNA harvested at 3 h was analyzed by QRT-PCR.

FIG. 7.

NaB and TPA activate EBV and KSHV lytic gene expression in dually infected BC-1 cells. (A) Southern blot of DNA from HH-B2 or BC-1 cells probed for EBV and KSHV DNA sequences. Probe for EBV DNA was BamHI W. Probe for KSHV DNA was KSR(339). (B) Northern blot for detection of EBV BRLF1 and BZLF1 mRNAs with the Z(301) probe. (C) Northern blot for detection of KSHV ORF50 mRNA with KSR(243) probe.

FIG. 8.

Differential effect of CHX on EBV BRLF1 and KSHV ORF50 mRNA levels in BC-1 cells treated with TPA. BC-1 cells were untreated (−) or treated (+) with TPA. CHX was added for the indicated durations, from 2 to 8 h before harvesting of RNA. RNA was prepared at 8 h. Northern blots were probed with Z(301) (A) or KSR(243) (B).

FIG. 9.

CHX inhibits expression of the EBV Rta and KSHV ORF50 proteins in BC-1 cells. Expression of EBV Rta and KSHV ORF50 proteins was analyzed by immunoblotting of cell extracts prepared in the same experiment (Fig. 8) in which the analysis of EBV BRLF1 and ORF50 mRNA by Northern blotting was studied. Cells were untreated or treated with the inducing agents, NaB or TPA. CHX was added to one aliquot of cells together with the inducing agent. Cell extracts were prepared 24 h after induction. Immunoblots were probed with monospecific polyclonal rabbit antibodies to the EBV Rta (A) or KSHV ORF50 (B) protein.

FIG. 10.

Quantitation of effects of CHX on EBV BRLF1 mRNA and KSHV ORF50 mRNA expression in BC-1 cells treated with NaB. BC-1 cells were treated with NaB for the indicated times. RNA was harvested at 1, 3, 5, and 8 h after treatment. One aliquot of cells received CHX simultaneously with NaB. The content of EBV BRLF1 mRNA (A) or KSHV ORF50 mRNA (B) was measured by QRT-PCR. The stimulation index (S.I.) was calculated relative to the content of RNA in untreated cells harvested at time zero.