Characterization of the gammaherpes virus specific tegument protein, pORF52, in t

伽马疱疹病毒特异性外皮蛋白 pORF52 的表征

• 批准号: 8194010
• 负责人: Melissa S Anderson
• 金额: $ 2.87万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-24 至 2013-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8194010
• 关键词: Address; Affinity Chromatography; Alanine; Antibodies; Bacterial Artificial Chromosomes; Binding; Biochemical; Biological Assay; Capsid; Capsid Proteins; Cell Culture Techniques; Cells; Cellular Structures; Centrifugation; Code; Complement; Confocal Microscopy; DNA biosynthesis; Data; Development; Fluorescence Microscopy; Goals; Homologous Gene; Homologous Protein; Human; Human Herpesvirus 8; Imaging Techniques; Immunoblotting; Immunofluorescence Microscopy; Immunoprecipitation; In Vitro; Individual; Infection; Investigation; Kaposi Sarcoma; Laboratories; Life Cycle Stages; Lymphoma; Lytic Phase; Macaca mulatta; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Microtubule Stabilization; Microtubules; Modeling; Monitor; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; Mutation; Open Reading Frames; Phenotype; Play; Production; Proteins; Publishing; Relative (related person); Rhadinovirus; Role; Scanning; Series; Staging; Structural Protein; Structure; Sucrose; Tertiary Protein Structure; Testing; Time; Transfection; Transmission Electron Microscopy; Tubulin; Viral; Viral Genome; Viral Proteins; Viral Structural Proteins; Virion; Virus; Work; base; effusion; extracellular; gamma-2 herpesvirus; gammaherpesvirus; insight; mutant; particle; protein expression; public health relevance; research study; tumor; viral DNA; viral rescue

DESCRIPTION (provided by applicant): Rhesus monkey rhadinovirus (RRV), a gammaherpesvirus, is a close viral relative of Kaposi's sarcoma (KS)- associated herpesvirus (KSHV or HHV8), the causative agent of three human malignancies, Kaposi's Sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. RRV is an effective model to study the structure and assembly of gammaherpesviruses since it is able to produce high levels of progeny viruses in culture. The insights gained from studying mechanisms governing virally induced cancers can extend to a wide variety of malignancies, helping in the development of new treatments and therapies. Previous data from our laboratory have shown that RRV contains five gammaherpesvirus specific proteins in its tegument (middle) layer and of those, three have unknown functions, including the protein encoded by open reading frame 52, pORF52. Our hypothesis is that pORF52 plays a critical role in the structure of virions (viral particles) and their assembly as well as a potential role in cellular egress via a direct or indirect interaction with critical infrastructural components of the cell known as microtubules (MTs). Our methods of investigation include utilization of a wild-type (WT) RRV and an ORF52 deleted ( 52) RRV bacterial artificial chromosomes (BAC), each of which contains the remainder of the viral genome. Introducing these BACs into cells capable of supporting viral production will allow us to monitor the viral life cycle in the presence or absence of pORF52 production. The first goal will be to determine whether pORF52 is necessary for RRV DNA replication, a step preceding and required for progeny viral production. Quantitative analysis of the levels of individual viral proteins within the BAC containing cells will then help determine the requirement of pORF52 in the production of the various building block proteins necessary for viral assembly. In addition to these analyses, various imaging techniques will generate complementary data, helping us to examine the structure of the individual particles, the gross level of their production within the cells, as well as their intracellular localization and stage of maturation. In addition, media from cells containing either WT or 52 BAC will be assessed for a) infectious virus and b) production of structural viral proteins. Our preliminary results indicate that pORF52 is necessary for production of virus. To test this, 52 BAC containing cells will be complemented with the introduction of exogenous pORF52 and subsequently examined for the presence of infectious virions as described above. Additionally, RRV pORF52 expression by itself leads to its co-localization with, and stabilization of microtubules. Included in the second half of this proposal is a series of experiments that will investigate this potential interaction using microtubule binding assays, immunofluorescence and confocal microscopy, and assays that directly assess physical interactions between these two components. Combined with a series of mutations strategically introduced into pORF52, these approaches will allow the identification of the regions within the protein essential for not only its MT interactions but also its role in the viral life cycle. PUBLIC HEALTH RELEVANCE: The insights gained from studying mechanisms governing virally induced cancers can extend to a wide variety of malignancies, helping in the development of new treatments and therapies. In this application, we propose to characterize the actions of one specific protein in the life cycle of a subfamily of tumor forming viruses called gammaherpesviruses. We hypothesize that this particular protein may play an essential role in the formation of new viral particles and, thus, would be an attractive candidate for targeted anti-viral therapy.

描述（由申请人提供）：恒河猴横流病毒（RRV）是一种伽玛疱疹病毒，是卡波西氏肉瘤（KS）相关疱疹病毒（KSHV或HHV8）的近亲病毒，卡波西氏肉瘤（KS）、原发性分泌性淋巴瘤和多中心Castleman病三种人类恶性肿瘤的病原体。RRV是研究γ疱疹病毒结构和组装的有效模型，因为它能够在培养中产生高水平的子代病毒。从研究病毒诱导癌症的机制中获得的见解可以扩展到各种各样的恶性肿瘤，有助于开发新的治疗方法。我们实验室先前的数据表明，RRV在其被膜（中间层）中含有5种γ疱疹病毒特异性蛋白，其中3种功能未知，包括开放阅读框52编码的蛋白pORF52。我们的假设是，pORF52在病毒粒子（病毒颗粒）的结构及其组装中起着关键作用，并通过与细胞的关键基础成分微管（MTs）的直接或间接相互作用在细胞出口中发挥潜在作用。我们的研究方法包括利用野生型（WT） RRV和ORF52缺失的（52）RRV细菌人工染色体（BAC），每条染色体都包含病毒基因组的剩余部分。将这些BACs引入能够支持病毒产生的细胞中，将使我们能够在存在或不存在pORF52产生的情况下监测病毒的生命周期。第一个目标将是确定pORF52是否是RRV DNA复制所必需的，这是子代病毒产生的先决条件。对含有BAC的细胞内单个病毒蛋白水平的定量分析将有助于确定在病毒组装所需的各种构建块蛋白的生产中对pORF52的需求。除了这些分析之外，各种成像技术将产生补充数据，帮助我们检查单个颗粒的结构，它们在细胞内产生的总体水平，以及它们在细胞内的定位和成熟阶段。此外，将对含有WT或52 BAC的细胞培养基进行评估，以确定a)感染性病毒和b)结构病毒蛋白的产生。我们的初步结果表明，pORF52是病毒产生所必需的。为了验证这一点，将52个含有BAC的细胞与引入外源性pORF52补充，随后检查感染性病毒粒子的存在，如上所述。此外，RRV pORF52本身的表达导致其与微管共定位和稳定。该提案的后半部分包括一系列实验，这些实验将使用微管结合测定、免疫荧光和共聚焦显微镜以及直接评估这两个组分之间物理相互作用的测定来研究这种潜在的相互作用。结合一系列战略性地引入pORF52的突变，这些方法将允许鉴定蛋白质内的区域，这些区域不仅对其MT相互作用至关重要，而且对其在病毒生命周期中的作用也至关重要。