调控宿主细胞周期是病毒感染的普遍规律。病毒通过调控细胞周期促进自身复制，同时影响细胞正常功能而致病。人巨细胞病毒（HCMV）是造成新生儿出生缺陷及在免疫缺陷病人中引起严重疾病的重要病原体。我们已经报道HCMV蛋白pUL117为病毒调控宿主细胞周期、促进病毒复制所必需。近期我们新发现了与pUL117特异结合的蛋白质Lin9和RbAp48，它们同属于MuvB复合物，MuvB与Rb/E2F等一起调控细胞周期基因的转录表达。由此我们推测pUL117通过与MuvB相互作用实现功能。本课题将在此基础上进一步探索1）pUL117和MuvB复合物相互作用在调控宿主细胞周期、促进病毒复制中的作用；2）与pUL117直接作用的MuvB复合物亚基及关键的相互作用位点；3）pUL117对MuvB复合物的组成、关键亚基翻译后修饰和功能的影响。本课题将有助于揭示HCMV调控细胞周期的分子机制，解析病毒复制和致病机理。

Deregulation of the host cell cycle is a common theme in virus infection, which helps creating a favorable cellular enviroment for viral replicatin. Meanwhile, cell cycle degregulation by virus infection often affects normal cellular functions,including cell proliferation and differetiation, leading to diseases. Human cytomegalovirus (HCMV) infection is one of the leading causes of birth defects. Its primary infection and reactivation also cause severe and even life threatening diseases in immunocomprimised individuals. We have reported that HCMV protein pUL117 was required for cell cylcle modulation and efficient virus replication. Our recent unpublished data indicated that pUL117 specifically interact with Lin9 and RbAp48. Both proteins are the components of the MuvB complex, which furhter forms complexes with Rb/E2F or b-myb, and subsequently controls gene transcription critical for cell cycle progression. Based on these data and knowledge,we hypothesize that pUL117 modulates the cell cycle and promotes viral replication through its interaction with the MuvB complex. In order to test our hypothesis, we will design experiments to address the following three key questions: 1)confirm that the interaction between pUL117 and the MuvB complex are important for its function; 2) identify the MuvB subunit that directly interacts with pUL117, and map out the key domains and motifs required for the interaction; 3) elucidate how pUL117 may affect the integrity, the post-translational modifications and the function of the MuvB complex. This study will provide new insights into the molecular mechanism of cell cycle deregulation by HCMV infection, help interperating the mechanism of HCMV replication and its pathogensis,and potentially lead to new approaches and identify novel drug targets to control CMV infection and its related diseases.

本研究通过免疫共沉淀结合免疫印迹（western blot）实验确定了与 pUL117 相互作用的MuvB 复合物亚基Lin9和Bmyb,并通过RNA干扰证明了敲低Lin9或Bmyb可以回复UL117突变病毒的晚期基因表达和复制缺陷。染色质免疫共沉淀(ChIP)实验表明pUL117能够结合在巨细胞病毒晚期基因UL99、UL32的启动子区域，是pUL117拮抗MuvB复合物、促进病毒晚期基因转录表达的可能机制。为了深入研究pUL117抑制宿主细胞周期进展的机制，本研究利用tet-on表达系统建立了pUL117诱导表达细胞系。在没有其他病毒蛋白存在的情况下，诱导表达pUL117可以在S期和G2/M期显著下调细胞周期蛋白Cyclin A和 Cyclin B的表达。反转录定量PCR实验表明pUL117对Cyclin A和Cyclin B的表达抑制发生在转录水平；双荧光素酶报告基因系统进一步确认了pUL117能够抑制cyclin A基因启动子活性。本研究工作还通过同源基因序列比对和点突变技术确定了pUL117氨基酸序列中介导抑制细胞周期蛋白表达的关键氨基酸。最后，敲低MuvB复合物中的Lin9能够回复UL117缺陷病毒感染细胞中细胞周期蛋白Cyclin B的表达抑制，明确了pUL117通过拮抗MuvB复合物抑制细胞周期蛋白表达。综上所述，本研究确认了人巨细胞病毒蛋白pUL117与MuvB复合物相互作用，通过拮抗其功能抑制S期和G2/M期细胞周期特定基因的表达，促进病毒晚期基因表达和病毒复制。