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.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...

Valproic Acid Antagonizes the Capacity of Other Histone Deacetylase Inhibitors To Activate the Epstein-Barr Virus Lytic Cycle

“…The efficiency of lytic induction is variable, is cell line dependent, and often requires the presence of another inducing agent with which these HDAC inhibitors act in synergy (33,40,61,101). These early studies of the switch from latency into the lytic phase of the EBV life cycle in cultured cells have evolved into attempts to manipulate EBV reactivation in a clinical setting.…”

“…Epigenetic modifiers, such as NaB, TSA, and AzaCdR, have been shown to reactivate latent viruses, such as the gammaherpesviruses EBV and KSHV, in a variety of cultured cell lines. However, the response to these agents is often cell line dependent (20,40,61,64,101).…”

“…Individual samples were assayed in triplicate. Primers either were designed using Primer3 (77) or have been described previously (26,101). The sequences of the primers are listed in Table 1.…”

Cellular defence or viral assist: the dilemma of HDAC6

“…This raises the possibility that inhibition of HDACs may reactivate latent proviruses. Indeed, HDAC6 inhibitors, as well as other HDACs, are subsequently found to reactivate HIV [141][142][143][144], EpsteinBarr virus [145][146][147], Kaposi's sarcoma-associated herpesvirus (KSHV) [148,149] and feline immunodeficiency virus [150].…”

RanBPM regulates Zta-mediated transcriptional activity in Epstein–Barr virus

“…During the switch from latency to the lytic cycle, EBV first expresses two immediate-early transcription factors, Rta and Zta, respectively encoded by BRLF1 and BZLF1. Rta and Zta regulate the expression of key EBV lytic genes (Chang et al, 2010;Chevallier-Greco et al, 1986;Giot et al, 1991;HolleyGuthrie et al, 1990;Kenney et al, 1989;Ye et al, 2007Ye et al, , 2010. Zta, a member of the bZip family (Farrell et al, 1989;Kouzarides et al, 1991), binds with Zta-responsive elements (ZREs) to activate the transcription of viral genes that are essential for lytic replication (Fixman et al, 1992) and also associates with TBP, TFIID and CBP to form a stable transcription initiation complex (Lieberman, 1994;Lieberman & Berk, 1991;Chen et al, 2001).…”