Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.
PLoS Pathog. 2010 Jun 24;6(6):e1000965. doi: 10.1371/journal.ppat.1000965.
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.
组蛋白去乙酰化在调节人类巨细胞病毒基因表达中起着关键作用。在本报告中，我们使用一种快速免疫分离与质谱联用的方法，鉴定了感染过程中候选的 HDAC1 相互作用蛋白。假定的相互作用蛋白包括多种人类巨细胞病毒编码蛋白。特别是，通过相互免疫沉淀证实了 pUL38 和 pUL29/28 与 HDAC1 的相互作用。HDAC1 存在于许多蛋白质复合物中，包括含有 HDAC1 的核小体重塑和去乙酰化酶蛋白复合物 NuRD。pUL38 和 pUL29/28 与 NuRD 的 MTA2 成分相关，并且 shRNA 介导的 NuRD 成分 RBBP4 和 CHD4 的敲低抑制了 HCMV 早期 RNA 和病毒 DNA 的积累；这表明 NuRD 复合物的多个成分对于 HCMV 复制是必需的。与 NuRD 元件在病毒复制过程中发挥积极作用一致，用去乙酰化酶抑制剂 Trichostatin A 处理感染细胞不能挽救 pUL29/28 或 pUL38 缺陷型病毒的生长。瞬时表达 pUL29/28 增强了 HCMV 主要早期启动子在报告基因检测中的活性，而与 pUL38 的表达无关。重要的是，pUL29/28 诱导主要早期报告基因活性需要功能性 NuRD 成分，这与 RBBP4 和 CHD4 敲低后感染细胞中早期 RNA 积累的抑制一致。我们提出 pUL29/28 修饰 NuRD 复合物以刺激早期 RNA 的积累。
