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.

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 疾病。 RRV是研究γ掌病毒的结构和组装的有效模型，因为它能够在培养物中产生高水平的后代病毒。从研究癌症癌症的机制中获得的见解可以扩展到各种恶性肿瘤，有助于开发新的治疗方法和疗法。我们实验室的先前数据表明，RRV在其tegument（中间）中包含五个伽马病毒特异性蛋白，其中三个具有未知功能，包括由开放阅读框架52，PORF52编码的蛋白质。我们的假设是，PORF52在病毒体（病毒颗粒）及其组件及其组件以及通过直接或间接相互作用与被称为微管（MTS）的关键基础结构组件的直接或间接相互作用在细胞出口中起关键作用。我们的研究方法包括利用野生型RRV（WT）RRV和ORF52删除（52）RRV细菌性人工染色体（BAC），每个染色体（BAC）都包含其余的病毒基因组。将这些BAC引入能够支持病毒生产的细胞中，将使我们能够在存在或不存在PORF52生产的情况下监测病毒生命周期。第一个目标是确定PORF52是否对于RRV DNA复制是必需的，这是前代病毒生产的前步骤和所需的步骤。然后，对包含BAC细胞中个体病毒蛋白水平的定量分析将有助于确定PORF52在生产病毒组装所需的各种构建块蛋白中的需求。除了这些分析外，各种成像技术还将产生互补数据，帮助我们检查单个颗粒的结构，细胞内的生产总水平，以及它们的细胞内定位和成熟阶段。此外，将评估包含WT或52 BAC的细胞的培养基，以评估A）传染病和B）产生结构性病毒蛋白的产生。我们的初步结果表明，PORF52对于生产病毒是必需的。为了测试这一点，含有52个BAC的细胞将与引入外源性Porf52的引入，并随后检查如上所述是否存在传染性病毒体。另外，RRV PORF52的表达本身会导致其与微管的共定位和稳定。该建议的下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研究这种潜在相互作用，并直接评估这两个组件之间的物理相互作用的测定法。结合一系列从策略上引入PORF52的突变，这些方法将允许鉴定蛋白质内部的MT相互作用所必需的区域，而且还可以鉴定其在病毒生命周期中的作用。 公共卫生相关性：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恶性肿瘤，有助于开发新的治疗和疗法。在此应用中，我们建议在肿瘤形成的称为Gammaherpesviruse的肿瘤病毒的生命周期中表征一种特定蛋白质的作用。我们假设该特定蛋白质可能在形成新的病毒颗粒的形成中起着至关重要的作用，因此将成为靶向抗病毒疗法的有吸引力的候选人。