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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.In this report we address the question of whether viral genes that regulate the lytic cycles of oncogenic human gammaherpesviruses behave with "immediate-early" kinetics upon reactivation from latency. Classical studies of the temporal pattern of expression of transcripts of bacteriophage T4 defined "early" genes, which were transcribed before DNA replication, and "late" genes, which were transcribed after viral DNA replication (41). Early genes were subdivided into "immediateearly" and "delayed-early" groups. Immediate-early transcripts appeared within 1 min after infection and were synthesized in the presence of chloramphenicol, an inhibitor of protein synthesis. Immediate-early, but not delayed-early, viral transcripts could be synthesized in vitro from DNA which was mechanically disrupted by shearing or sonication. This result implied that immediate-early genes we...

Epstein-Barr virus (EBVOncogenic human herpesviruses, such as Epstein-Barr virus (EBV), manifest two distinct lifestyles: latency, a state of limited viral gene expression, and lytic replication, which ultimately leads to production of virions. The switch between latency and productive lytic infection can be manipulated in cell culture. Lymphoid cell lines are unique experimental systems with which to study physiologic and molecular mechanisms underlying the transition between the two life cycles. The switch between viral latency and lytic replication is a biologically interesting and potentially tractable example of the combinatorial control of eukaryotic gene expression. Groups of viral and cellular effector molecules, some of which are transcription factors, exert both positive and negative control on the expression and the activity of two virally encoded proteins, ZEBRA and Rta, both of which act as transcription factors and replication proteins. Epigenetic control of viral and cellular gene expression, through chromatinization and DNA methylation, may also play roles in the latency-to-lytic cycle transition. The latency-to-lytic cycle switch has obvious implications for pathogenesis. While latency may be the predominant state of the life cycle in cellular reservoirs and in virus-associated cancers, the viruses must replicate lytically in order to be transmitted between cells and among individuals. Manipulation of the latency-to-lytic cycle switch has been investigated as a potential oncolytic strategy (11,28,36,43).The latency-to-lytic switch can be envisioned to be composed of two distinct complex sets of events: upstream events lead to the expression of the EBV lytic cycle activator genes BZLF1 and BRLF1, which encode ZEBRA and Rta, and downstream events involve the effects of ZEBRA and Rta and their target genes on viral and cellular gene expression, DNA replication, and viral and cellular behavior in general. Upstream events can be initiated in cell culture by the addition of certain inducing stimuli which presumably mimic asyet poorly characterized physiologic stimuli that trigger the latency-to-lytic cycle switch in vivo. A partial list of stimuli that can activate the latency-to-lytic switch in cultured B-cell lines includes phorbol esters (47), which are protein kinase C (PKC) agonists; sodium butyrate (NaB) (26) and trichostatin A (TSA) (46), which are histone deacetylase inhibitors (HDACi); 5-aza-2-Ј-deoxycytidine (AzaCdR) (4), which is a DNA methyltransferase inhibitor; and anti-immunoglobulin G (anti-IgG), which activates the B-cell antigen receptor (40).Inspection of this list of inducing stimuli, which are thought to operate by different modes of action, leads to the conclusion that upstream events are likely to activate different pathways which lead to BZLF1 and BRLF1 expression. These pathways may or may not converge on a final common event. Further complexity in understanding the upstream events is evident from the observation that not all cell/virus systems respond to the same inducing sti...