Activation and repression of Epstein-Barr Virus and Kaposi's sarcoma-associated herpesvirus lytic cycles by short- and medium-chain fatty acids.

The lytic cycles of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are induced in cell culture by sodium butyrate (NaB), a short-chain fatty acid (SCFA) histone deacetylase (HDAC) inhibitor. Valproic acid (VPA), another SCFA and an HDAC inhibitor, induces the lytic cycle of KSHV but blocks EBV lytic reactivation. To explore the hypothesis that structural differences between NaB and VPA account for their functional effects on the two related viruses, we investigated the capacity of 16 structurally related short- and medium-chain fatty acids to promote or prevent lytic cycle reactivation. SCFAs differentially affected EBV and KSHV reactivation. KSHV was reactivated by all SCFAs that are HDAC inhibitors, including phenylbutyrate. However, several fatty acid HDAC inhibitors, such as isobutyrate and phenylbutyrate, did not reactivate EBV. Reactivation of KSHV lytic transcripts could not be blocked completely by any fatty acid tested. In contrast, several medium-chain fatty acids inhibited lytic activation of EBV. Fatty acids that blocked EBV reactivation were more lipophilic than those that activated EBV. VPA blocked activation of the BZLF1 promoter by NaB but did not block the transcriptional function of ZEBRA. VPA also blocked activation of the DNA damage response that accompanies EBV lytic cycle activation. Properties of SCFAs in addition to their effects on chromatin are likely to explain activation or repression of EBV. We concluded that fatty acids stimulate the two related human gammaherpesviruses to enter the lytic cycle through different pathways. Importance: Lytic reactivation of EBV and KSHV is needed for persistence of these viruses and plays a role in carcinogenesis. Our direct comparison highlights the mechanistic differences in lytic reactivation between related human oncogenic gammaherpesviruses. Our findings have therapeutic implications, as fatty acids are found in the diet and produced by the human microbiota. Small-molecule inducers of the lytic cycle are desired for oncolytic therapy. Inhibition of viral reactivation, alternatively, may prove useful in cancer treatment. Overall, our findings contribute to the understanding of pathways that control the latent-to-lytic switch and identify naturally occurring molecules that may regulate this process.

Valproic acid antagonizes the capacity of other histone deacetylase inhibitors to activate the Epstein-barr virus lytic cycle.

Diverse stimuli reactivate the Epstein-Barr virus (EBV) lytic cycle in Burkitt lymphoma (BL) cells. In HH514-16 BL cells, two histone deacetylase (HDAC) inhibitors, sodium butyrate (NaB) and trichostatin A (TSA), and the DNA methyltransferase inhibitor azacytidine (AzaCdR) promote lytic reactivation. Valproic acid (VPA), which, like NaB, belongs to the short-chain fatty acid class of HDAC inhibitors, fails to induce the EBV lytic cycle in these cells. Nonetheless, VPA behaves as an HDAC inhibitor; it causes hyperacetylation of histone H3 (J. K. Countryman, L. Gradoville, and G. Miller, J. Virol. 82:4706-4719, 2008). Here we show that VPA blocked the induction of EBV early lytic proteins ZEBRA and EA-D in response to NaB, TSA, or AzaCdR. The block in lytic activation occurred prior to the accumulation of BZLF1 transcripts. Reactivation of EBV in Akata cells, in response to anti-IgG, and in Raji cells, in response to tetradecanoyl phorbol acetate (TPA), was also inhibited by VPA. MS-275 and apicidin, representing two additional classes of HDAC inhibitors, and suberoylanilide hydroxamic acid (SAHA) reactivated EBV in HH514-16 cells; this activity was also inhibited by VPA. Although VPA potently blocked the expression of viral lytic-cycle transcripts, it did not generally block the transcription of cellular genes and was not toxic. The levels and kinetics of specific cellular transcripts, such as Stat3, Frmd6, Mad1, Sepp1, c-fos, c-jun, and egr1, which were activated by NaB and TSA, were similar in HH514-16 cells treated with VPA. When combined with NaB or TSA, VPA did not inhibit the activation of these cellular genes. Changes in cellular gene expression in response to VPA, NaB, or TSA were globally similar as assessed by human genome arrays; however, VPA selectively stimulated the expression of some cellular genes, such as MEF2D, YY1, and ZEB1, that could repress the EBV lytic cycle. We describe a novel example of functional antagonism between HDAC inhibitors.

Upregulation of STAT3 marks Burkitt lymphoma cells refractory to Epstein-Barr virus lytic cycle induction by HDAC inhibitors.

A fundamental problem in studying the latent-to-lytic switch of Epstein-Barr virus (EBV) and the viral lytic cycle itself is the lack of a culture system fully permissive to lytic cycle induction. Strategies to target EBV-positive tumors by inducing the viral lytic cycle with chemical agents are hindered by inefficient responses to stimuli. In vitro, even in the most susceptible cell lines, more than 50% of cells latently infected with EBV are refractory to induction of the lytic cycle. The mechanisms underlying the refractory state are not understood. We separated lytic from refractory Burkitt lymphoma-derived HH514-16 cells after treatment with an HDAC inhibitor, sodium butyrate. Both refractory- and lytic-cell populations responded to the inducing stimulus by hyperacetylation of histone H3. However, analysis of host cell gene expression showed that specific cellular transcripts Stat3, Fos, and interleukin-8 (IL-8) were preferentially upregulated in the refractory-cell population, while IL-6 was upregulated in the lytic population. STAT3 protein levels were also substantially increased in refractory cells relative to untreated or lytic cells. This increase in de novo expression resulted primarily in unphosphorylated STAT3. Examination of single cells revealed that high levels of STAT3 were strongly associated with the refractory state. The refractory state is manifest in a unique subpopulation of cells that exhibits different cellular responses than do lytic cells exposed to the same stimulus. Identifying characteristics of cells refractory to lytic induction relative to cells that undergo lytic activation will be an important step in developing a better understanding of the regulation of the EBV latent to lytic switch.

Valpromide Inhibits Lytic Cycle Reactivation of Epstein-Barr Virus.

UNLABELLED: Reactivation of Epstein-Barr virus (EBV) from latency into the lytic phase of its life cycle allows the virus to spread among cells and between hosts. Valproic acid (VPA) inhibits initiation of the lytic cycle in EBV-infected B lymphoma cells. While VPA blocks viral lytic gene expression, it induces expression of many cellular genes, because it is a histone deacetylase (HDAC) inhibitor. Here we show, using derivatives of VPA, that blockade of EBV reactivation is separable from HDAC inhibition. Valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented expression of two EBV genes, BZLF1 and BRLF1, that mediate lytic reactivation. VPM also inhibited expression of a viral late gene, but not early genes, when BZLF1 was exogenously expressed. Unlike VPA, VPM did not activate lytic expression of Kaposi's sarcoma-associated herpesvirus. Expression of cellular immediate-early genes, such as FOS and EGR1, is kinetically upstream of the EBV lytic cycle. VPM did not activate expression of these cellular immediate-early genes but decreased their level of expression when induced by butyrate, an HDAC inhibitor. VPM did not alter expression of several other cellular immediate-early genes, including STAT3, which were induced by the HDAC inhibitors in cells refractory to lytic induction. Therefore, VPM selectively inhibits both viral and cellular gene expression. VPA and VPM represent a new class of antiviral agents. The mechanism by which VPA and VPM block EBV reactivation may be related to their anticonvulsant activity. IMPORTANCE: Epstein-Barr virus, (EBV), a human tumor virus, establishes a life-long latent infection. Reactivation of EBV into the lytic phase of its life cycle allows the virus to spread. Previously, we showed that EBV reactivation was blocked by valproic acid (VPA), an inhibitor of cellular histone deacetylases (HDACs). VPA alters the expression of thousands of cellular genes. In this study, we demonstrate that valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented initiation of the EBV lytic cycle. VPA induced lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV), but VPM did not. Unlike VPA, VPM did not activate cellular immediate-early gene expression. VPM is a new type of antiviral agent. VPM will be useful in probing the mechanism of EBV lytic reactivation and may have therapeutic application.

High-throughput sequencing of PCR products tagged with universal primers using 454 life sciences systems.

This unit describes a method to convert PCR products (amplicons) flanked by universal M13 primers into a library for use on all 454 Sequencing Systems (454 Life Sciences, a Roche Company). This is especially useful for simultaneous sequencing and analysis of large numbers of amplicons or for the detection of minor variations within the amplified products. The method described here involves preparing a library of DNA with specific primers containing adaptor sequences recognized by the GS Junior System sequencing process. The data from the sequencing run are processed and analyzed by 454 Life Sciences software. This approach allows for multiplexing of a large number of amplicons to streamline processing and analysis. Any pre-existing universally tagged amplicon, primer set, or plasmid with M13 sequences flanking the cloning site can be used in this protocol.

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.