FOLLOWING THE FATE OF HCMV GENE PRODUCTS IN HOST CELLS

追踪 HCMV 基因产物在宿主细胞中的命运

基本信息

批准号：
8169121
负责人：
THOMAS E SHENK
金额：
$ 4.65万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8169121
关键词：
Adult Age-Years Apoptosis Cells Cellular Stress Characteristics Collaborations Complex Computer Retrieval of Information on Scientific Projects Database Cytomegalovirus Cytomegalovirus Infections Epitopes Exhibits Funding Geographic Locations Grant Herpesviridae Infection Institution Laboratories Manuscripts Mass Spectrum Analysis Microbe Pathway interactions Protein Biosynthesis Proteins Publishing Research Research Personnel Resources Route Signal Transduction Source Staging Stress T-Lymphocyte TSC1 gene TSC1/2 gene TSC2 gene Tuberous Sclerosis Tuberous sclerosis protein complex Tumor Suppressor Proteins United States United States National Institutes of Health Universities Viral Viral Genome Viral Proteins Virus Replication Work biological adaptation to stress cell growth insight mTOR protein prevent protein complex response socioeconomics

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytomegalovirus is widely spread throughout all geographic locations and socioeconomic group and infects 85% of adults in United States by 40 years of age. Our aim is to study the interactions of human cytomegalovirus with cellular proteins, and gain insights into cellular pathways modulated by herpesviruses. This study was initiated one and a half years ago in collaboration with the laboratory of Tom Shenk at Princeton University. Since then, we have isolated a variety of Protein A- or GFP-tagged HCMV viral proteins identifying their host and viral interacting partners at various stages of infections. During this last year, we have studied the interacting partners of several viral proteins, including UL83, UL121, UL38, UL99, and US22. Each of these studies highlighted new ways that HCMV utilizes to manipulate and take over the cell. Human cytomegalovirus proteins alter host cells to favor virus replication. These viral proteins include pUL38, which prevents apoptosis. To characterize the mode of action of pUL38, we modified the viral genome to encode an epitope-tagged pUL38 and used rapid immunoaffinity purification to isolate pUL38-interacting host proteins, which were then identified by mass spectrometry. One of the cellular proteins identified was TSC2, a constituent of the tuberous sclerosis tumor suppressor protein complex (TSC1/2). TSC1/2 integrates stress signals and regulates the mammalian target of rapamycin complex 1 (mTORC1), a protein complex that responds to stress by limiting protein synthesis and cell growth. We showed that pUL38 interacts with TSC1 and TSC2 in cells infected with wild-type cytomegalovirus. Furthermore, TSC1/2 failed to regulate mTORC1 in cells expressing pUL38, and these cells exhibited the enlarged size characteristic of cytomegalovirus infection. Thus, pUL38 supports virus replication at least in part by blocking cellular responses to stress. A manuscript describing this work has been published: Human cytomegalovirus protein UL38 inhibits host cell stress responses by antagonizing the tuberous sclerosis protein complex. Moorman NJ, Cristea IM, Terhune SS, Rout MP, Chait BT, Shenk T. Cell Host Microbe. 2008 3(4):253-62. Most recently, we have prepared two additional manuscrpts in which immunoisolation mass spectrometry was used to gain clues as to the function of viral proteins in their hosts (see below)

该副本是利用众多研究子项目之一由NIH/NCRR资助的中心赠款提供的资源。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是对于中心，这不一定是调查员的机构。巨细胞病毒广泛分布在所有地理位置和社会经济群体中，并在40岁时感染了美国85％的成年人。我们的目的是研究人类巨细胞病毒与细胞蛋白的相互作用，并获得对由疱疹病毒调节的细胞途径的见解。这项研究是在半年前与普林斯顿大学汤姆·申克（Tom Shenk）的实验室合作的。从那时起，我们分离了各种蛋白A-或GFP标记的HCMV病毒蛋白，以识别其宿主和病毒相互作用伴侣在感染的各个阶段。去年，我们研究了几种病毒蛋白的相互作用伙伴，包括UL83，UL121，UL38，UL99和US22。这些研究中的每一个都强调了HCMV用来操纵和接管细胞的新方法。人巨细胞病毒蛋白改变宿主细胞以有利于病毒复制。这些病毒蛋白包括PUL38，可防止凋亡。为了表征PUL38的作用方式，我们修改了病毒基因组以编码表位标记的PUL38并使用快速的免疫亲和力纯化来分离PUL38相互作用的宿主蛋白，然后通过质谱法鉴定出来。鉴定出的细胞蛋白之一是TSC2，TSC2是结节性硬化肿瘤抑制蛋白复合物（TSC1/2）的组成部分。 TSC1/2整合了应力信号并调节雷帕霉素复合物1（MTORC1）的哺乳动物靶标，这是一种蛋白质复合物，通过限制蛋白质的合成和细胞生长来响应胁迫。我们表明，在感染野生型巨细胞病毒的细胞中，PUL38与TSC1和TSC2相互作用。此外，TSC1/2未能调节表达PUL38的细胞中的MTORC1，并且这些细胞表现出巨型巨细胞病毒感染的大小特征。因此，PUL38至少部分通过阻止细胞对胁迫的反应至少部分支持病毒复制。描述这项工作的手稿已经发表：人巨细胞病毒蛋白UL38通过拮抗结节性硬化蛋白复合物，抑制宿主的细胞应激反应。 Moorman NJ，Cristea IM，Terhune SS，Rout MP，Chait BT，Shenk T.细胞宿主微生物。 2008 3（4）：253-62。最近，我们已经准备了另外两种手法，其中使用了免疫分散质谱法来获得有关宿主中病毒蛋白功能的线索（见下文）