Human cytomegalovirus UL29/28 protein interacts with components of the NuRD complex which promote accumulation of immediate-early RNA.
Human cytomegalovirus UL29/28 protein interacts with components of the NuRD complex which promote accumulation of immediate-early RNA.
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DOI：
10.1371/journal.ppat.1000965
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发表时间：
2010-06-24
期刊：
影响因子：
6.7
通讯作者：
Shenk T
中科院分区：
文献类型：
--
作者：
Terhune SS;Moorman NJ;Cristea IM;Savaryn JP;Cuevas-Bennett C;Rout MP;Chait BT;Shenk T
组蛋白去乙酰化在人巨细胞病毒基因表达调控中起着关键作用。在这份报告中，我们使用一种结合质谱仪的表位标记蛋白的快速免疫分离方法，在感染背景下鉴定了候选的HDAC1相互作用蛋白。推测的相互作用因素包括多种人类巨细胞病毒编码蛋白。特别是，pUL38和pUL29/28与HDAC1的相互作用通过相互免疫沉淀得到证实。HDAC1存在于许多蛋白质复合体中，包括含有HDAC1的核小体重塑和脱乙酰酶蛋白质复合体NuRD。与NuRD的MTA2组分相关的pUL38和pUL29/28，以及shRNA介导的对NuRD的RBBP4和CHD4组分的敲除抑制了HCMV即刻早期RNA和病毒DNA的积累，这表明NuRD复合体的多个组成部分是有效复制HCMV所必需的。与NuRD元件在病毒复制过程中的积极作用一致，pUL29/28或pUL38缺陷病毒的生长不能通过去乙酰化酶抑制剂曲古菌素A处理感染细胞来挽救。瞬时表达pUL29/28在报告实验中增强了HCMV主要即刻早期启动子的活性，而与pUL38表达无关。重要的是，pUL29/28诱导主要的即刻早期报告活性需要有功能的NuRD成分，这与在RBBP4和CHD4被击倒后抑制感染细胞内的即刻早期RNA积累一致。我们建议pUL29/28修改NuRD复合体，以刺激立即-早期RNA的积累。调控基因表达的一个关键事件涉及核心组蛋白乙酰化状态的变化。调节是通过组蛋白乙酰转移酶添加乙酰基和脱乙酰酶去除部分之间的平衡来完成的。这些变化在调节细胞分化和增殖方面是必不可少的，同样，干扰会导致包括癌症在内的各种病理变化。此外，这些关键调节因子是疱疹病毒的靶标，以确保宿主在一生中持续感染。在疱疹病毒人类巨细胞病毒(HCMV)的病例中，组蛋白乙酰化的变化与感染的潜伏期和急性期的选择有关。我们使用了一种聚焦的蛋白质组学方法来鉴定在急性巨细胞病毒感染过程中与组蛋白脱乙酰基酶1(HDAC1)蛋白相互作用和调节的蛋白质。我们的研究确定了大量的细胞和病毒蛋白，包括HCMV pUL29/28。这种蛋白结合到核小体重塑和脱乙酰酶复合体的组件NuRD，以及功能性NuRD组件是表达和感染HCMV所必需的。我们的研究展示了一个新的工具来研究宿主和病原体的相互作用，同时也为HDAC1在HCMV复制过程中的复杂调控提供了新的见解。
Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs. A key event in regulating gene expression involves changes in the acetylation status of core histones. Regulation is accomplished by a balance between the addition of acetyl groups by histone acetyltransferase enzymes and removal of the moieties by deacetylases. These changes are essential in regulating cellular differentiation and proliferation and, likewise, disruption results in a variety of pathologies, including cancer. In addition, these key regulators are targeted by herpesviruses to ensure persistent infection during the life of the host. In the case of the herpesvirus human cytomegalovirus (HCMV), changes in histone acetylation have been implicated in the choice between latent and acute phases of infection. We have used a focused proteomics approach to identify proteins that are interacting with and regulating the histone deacetylase 1 (HDAC1) protein during acute cytomegalovirus infection. Our studies identified numerous cellular and viral proteins including HCMV pUL29/28. This protein bound to components of the nucleosome remodeling and deacetylase complex, NuRD, and functional NuRD components were necessary for HCMV gene expression and infection. Our study demonstrates a new tool for studying host-pathogen interactions as well as provides new insights into the complex regulation of HDAC1 during HCMV replication.
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