Cellular immediate-early gene expression occurs kinetically upstream of Epstein-Barr virus bzlf1 and brlf1 following cross-linking of the B cell antigen receptor in the Akata Burkitt lymphoma cell line.

The Epstein-Barr virus (EBV) lytic activator genes bzlf1 and brlf1 are conventionally referred to as immediate-early (IE) genes. However, previous studies showed that the earliest expression of these genes was blocked by cycloheximide when the EBV lytic cycle was induced by histone deacetylase (HDAC) inhibitors and protein kinase C agonists. Anti-IgG activates a complex signal transduction pathway that leads to EBV lytic activation in the Akata cell line. Here we demonstrate that in Akata cells, where lytic cycle activation occurs very rapidly after anti-IgG treatment, de novo protein synthesis is also required for induction of bzlf1 and brlf1 expression. New protein synthesis is required up to 1.25 h after application of anti-IgG; bzlf1 and brlf1 mRNAs can be detected 1.5 h after anti-IgG. Five cellular IE genes were shown to be expressed by 1 h after addition of anti-IgG, and their expression preceded that of bzlf1 and brlf1. These include early growth response genes (egr1, egr2, and egr3) and nuclear orphan receptors (nr4a1 and nr4a3). These genes were activated by anti-IgG treatment of Akata cells with and without the EBV genome; therefore, their expression was not dependent on expression of any EBV gene product. EGR1, EGR2, and EGR3 proteins were kinetically upstream of ZEBRA and Rta proteins. Expression of EGR1, ZEBRA, and Rta proteins were inhibited by bisindolylmaleimide X, a selective inhibitor of PKC. The findings suggest a revised model in which the signal transduction cascade activated by cross-linking of the B cell receptor induces expression of cellular IE genes, such as early growth response and nuclear orphan receptor genes, whose products, in turn, regulate bzlf1 and brlf1 expression.

Stimulus duration and response time independently influence the kinetics of lytic cycle reactivation of Epstein-Barr virus.

Epstein-Barr virus (EBV) can be reactivated from latency into the lytic cycle by many stimuli believed to operate by different mechanisms. Cell lines containing EBV differ in their responses to inducing stimuli, yet all stimuli require de novo protein synthesis (44). A crucial step preliminary to identifying these proteins and determining when they are required is to measure the duration of stimulus and response time needed for activation of expression of EBV BRLF1 and BZLF1, the earliest viral indicators of reactivation. Here we show, with four EBV-containing cell lines that respond to different inducing agents, that stimuli that are effective at reactivating EBV can be divided into two main groups. The histone deacetylase inhibitors sodium butyrate and trichostatin A require a relatively long period of exposure, from 2 to 4 h or longer. Phorbol esters, anti-immunoglobulin G (anti-IgG), and, surprisingly, 5-aza-2'-deoxycytidine require short exposures of 15 min or less. The cell/virus background influences the response time. Expression of the EBV BZLF1 and BRLF1 genes can be detected before 2 h in Akata cells treated with anti-IgG, but both long- and short-duration stimuli required 4 or more hr to activate BZLF1 and BRLF1 expression in HH514-16, Raji, or B95-8 cells. Thus, stimulus duration and response time are independent variables. Neither stimulus duration nor response time can be predicted by the number of cells activated into the lytic cycle. These experiments shed new light on the earliest events leading to lytic cycle reactivation of EBV.

Histone hyperacetylation occurs on promoters of lytic cycle regulatory genes in Epstein-Barr virus-infected cell lines which are refractory to disruption of latency by histone deacetylase inhibitors.

Activation of the Epstein-Barr virus (EBV) lytic cycle is mediated through the combined actions of ZEBRA and Rta, the products of the viral BZLF1 and BRLF1 genes. During latency, these two genes are tightly repressed. Histone deacetylase inhibitors (HDACi) can activate viral lytic gene expression. Therefore, a widely held hypothesis is that Zp and Rp, the promoters for BZLF1 and BRLF1, are repressed by chromatin and that hyperacetylation of histone tails, by allowing the access of positively acting factors, leads to transcription of BZLF1 and BRLF1. To investigate this hypothesis, we used chromatin immunoprecipitation (ChIP) to examine the acetylation and phosphorylation states of histones H3 and H4 on Zp and Rp in three cell lines, Raji, B95-8, and HH514-16, which differ in their response to EBV lytic induction by HDACi. We studied the effects of three HDACi, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA). We also examined the effects of tetradecanoyl phorbol acetate (TPA) and 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, on histone modification. In Raji cells, TPA and NaB act synergistically to activate the EBV lytic cycle and promote an increase in histone H3 and H4 acetylation and phosphorylation at Zp and Rp. Surprisingly, however, when Raji cells were treated with NaB or TSA, neither of which is sufficient to activate the lytic cycle, an increase of comparable magnitude of hyperacetylated and phosphorylated histone H3 at Zp and Rp was observed. In B95-8 cells, NaB inhibited lytic induction by TPA, yet NaB promoted hyperacetylation of H3 and H4. In HH514-16 cells, NaB and TSA strongly activated the EBV lytic cycle and caused hyperacetylation of histone H3 on Zp and Rp. However, when HH514-16 cells were treated with VPA, lytic cycle mRNAs or proteins were not induced, although histone H3 was hyperacetylated as measured by immunoblotting or by ChIP on Zp and Rp. Taken together, our data suggest that open chromatin at EBV BZLF1 and BRLF1 promoters is not sufficient to activate EBV lytic cycle gene expression.

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.

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.

Protein kinase C-independent activation of the Epstein-Barr virus lytic cycle.

The protein kinase C (PKC) pathway has been considered to be essential for activation of latent Epstein-Barr virus (EBV) into the lytic cycle. The phorbol ester tetradecanoyl phorbol acetate (TPA), a PKC agonist, is one of the best understood activators of EBV lytic replication. Zp, the promoter of the EBV immediate-early gene BZLF1, whose product, ZEBRA, drives the lytic cycle, contains several phorbol ester response elements. We investigated the role of the PKC pathway in lytic cycle activation in prototype cell lines that differed dramatically in their response to inducing agents. We determined whether PKC was involved in lytic cycle induction by histone deacetylase (HDAC) inhibitors. Consistent with prevailing views, B95-8 cells were activated into the lytic cycle by the phorbol ester TPA, via a PKC-dependent mechanism. B95-8 was not inducible by HDAC inhibitors such as n-butyrate and trichostatin A (TSA). Bisindolylmaleimide I, a selective PKC inhibitor, blocked lytic cycle activation in B95-8 cells in response to TPA. In marked contrast, in HH514-16 cells, the immediate-early promoters Zp and Rp were simultaneously activated by the HDAC inhibitors; TPA by itself failed to activate lytic gene expression. Inhibition of PKC activity by bisindolylmaleimide I did not block lytic cycle activation in HH514-16 cells by n-butyrate or TSA. In an extensive exploration of the mechanism underlying these different responses we found that the variable role of the PKC pathway in the two cell lines could not be accounted for by significant polymorphisms in the promoters of the immediate-early genes, by differences in the start sites of immediate-early gene transcription, or by differences in the nucleosomal organization of EBV DNA in the region of Zp or Rp. While B95-8 cells contained more total PKC activity than did HH514-16 cells in an in vitro assay, another EBV-transformed marmoset lymphoblastoid cell line, FF41, in which the lytic cycle was not inducible by TPA, contained comparably high levels of PKC activity. Moreover, two marmoset lymphoblastoid cells lines in which the lytic cycle could not be triggered by TPA maintained the same profile of EBV latency proteins as B95-8 cells. Thus, the profile of EBV latency proteins did not account for susceptibility to induction by PKC agonists. PKC activation is neither obligatory nor sufficient for the switch between latency and lytic cycle gene expression of EBV in many cell backgrounds. Lytic cycle induction by HDAC inhibitors proceeds by a PKC-independent mechanism.