Lu Fang, Zhou Jing, Wiedmer Andreas, Madden Kevin, Yuan Yan, Lieberman Paul M
The Wistar Institute. Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Virol. 2003 Nov;77(21):11425-35. doi: 10.1128/jvi.77.21.11425-11435.2003.
The switch from latent to lytic infection of Kaposi's sarcoma-associated herpesvirus is initiated by the immediate early transcriptional activator protein Rta/open reading frame 50 (ORF50). We examined the transcriptional regulation of the ORF50 core promoter in response to lytic cycle stimulation. We show that the ORF50 promoter is highly responsive to sodium butyrate (NaB) and trichostatin A (TSA), two chemicals known to inhibit histone deacetylases. The NaB and TSA responsive element was mapped to a 70-bp minimal promoter containing an essential GC box that binds Sp1/Sp3 in vitro and in vivo. Micrococcal nuclease mapping studies revealed that a nucleosome is positioned over the transcriptional initiation and the Sp1/3 binding sites. Stimulation with NaB or TSA increased histone acetylation and restriction enzyme accessibility of the ORF50 promoter transcription initiation site. Chromatin immunoprecipitation assay was used to demonstrate that the ORF50 promoter is associated with several different histone deacetylase proteins (including HDAC1, 5, and 7) in latently infected cells. NaB treatment led to the rapid association of Ini1/Snf5, a component of the Swi/Snf family of chromatin remodeling proteins, with the ORF50 promoter. Ectopic expression of the CREB-binding protein (CBP) histone acetyltransferase (HAT) stimulated plasmid-based ORF50 transcription in a HAT-dependent manner, suggesting that CBP recruitment to the ORF50 promoter can be an initiating event for transcription and viral reactivation. Together, these results suggest that remodeling of a stably positioned nucleosome at the transcriptional initiation site of ORF50 is a regulatory step in the transition from latent to lytic infection.
卡波西肉瘤相关疱疹病毒从潜伏感染转变为裂解感染是由即刻早期转录激活蛋白Rta/开放阅读框50（ORF50）启动的。我们研究了ORF50核心启动子在裂解周期刺激下的转录调控。我们发现ORF50启动子对丁酸钠（NaB）和曲古抑菌素A（TSA）高度敏感，这两种化学物质已知可抑制组蛋白脱乙酰酶。NaB和TSA反应元件被定位到一个70 bp的最小启动子，该启动子包含一个必需的GC框，在体外和体内均可与Sp1/Sp3结合。微球菌核酸酶图谱研究表明，一个核小体位于转录起始位点和Sp1/3结合位点上方。用NaB或TSA刺激可增加组蛋白乙酰化以及ORF50启动子转录起始位点的限制酶可及性。染色质免疫沉淀试验用于证明在潜伏感染的细胞中，ORF50启动子与几种不同的组蛋白脱乙酰酶蛋白（包括HDAC1、5和7）相关。NaB处理导致染色质重塑蛋白Swi/Snf家族的一个组分Ini1/Snf5迅速与ORF50启动子结合。CREB结合蛋白（CBP）组蛋白乙酰转移酶（HAT）的异位表达以HAT依赖的方式刺激基于质粒的ORF50转录，这表明CBP募集到ORF50启动子可能是转录和病毒重新激活的起始事件。总之，这些结果表明，在ORF50转录起始位点对稳定定位的核小体进行重塑是从潜伏感染转变为裂解感染过程中的一个调控步骤。
