Engineering Herpesviruses using Synthetic Genomics

使用合成基因组学改造疱疹病毒

• 批准号: 8893391
• 负责人: PRASHANT J DESAI
• 金额: $ 27.21万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8893391
• 关键词: B-Lymphocytes; Bacterial Genome; Basic Science; Biological; Biological Assay; Biology; Cells; Chemicals; Code; Complex; DNA; DNA Viruses; Engineering; Episome; Escherichia coli; Future; Genetic; Genome; Genome engineering; Genomic approach; Genomics; Goals; Herpesviridae; Herpesvirus 1; Human Herpesvirus 4; Immunoglobulin G; Individual; Institutes; Laboratories; Lytic; Lytic Virus; Mammalian Cell; Measures; Methods; Microbe; Modeling; Molecular Biology; Molecular Genetics; One-Step dentin bonding system; Outcome; Pharmaceutical Preparations; Plasmids; Process; Production; Property; Proteins; Reagent; Scientist; Technology; Testing; Time; Transgenes; Viral Genome; Virion; Virus; Yeasts; base; crosslink; established cell line; gammaherpesvirus; genetic manipulation; genome sequencing; high risk; member; mutant; pathogen; prototype; public health relevance; reactivation from latency; recombinase; reconstitution; synthetic biology; time use; tool

 DESCRIPTION (provided by applicant): Herpesviruses are large DNA viruses whose genomes have the coding potential of in excess of 100 gene products. Historically, genetic manipulation of these large genomes was feasible for only a subset of these viruses. Subsequently, many of the herpesvirus genomes were cloned into BAC plasmids, which significantly advanced the technologies of genome engineering in an E.coli host and the successful reconstitution of infectious virus in the appropriate host cell. In this application, we propose to use synthetic biology to first build a wild-type clone of the Epstein-Barr Virus (EBV) genome and then reconstitute the infectious virus. Herpes simplex virus type 1 (HSV-1) will be used as a model to first test and optimize the assembly method. The successful outcome of this synthetic biology approach will have a transformational impact on the ability to synthetically clone and manipulate any herpesvirus genome. In addition, this approach offers a paradigm to help understand the molecular genetics and biology of emerging pathogens. Specific Aim 1. Use synthetic genomics methods to assemble an infectious genome of EBV. In this aim, we will clone the EBV genome, strain Akata, using synthetic genomics. Our approach will be based on recent advances made in this field by members of the J. Craig Venter Institute (JCVI) team that created the first synthetic microbe. The JCVI lab has developed methods that enable the assembly of large DNA fragments, ranging from hundred kilobases to megabase size genomes. This team will use these methods to assemble an infectious clone of EBV and at the same time, use the more "tractable" HSV-1 genome assembly to optimize and refine synthetic genomics methods. Specific Aim 2. Establish a cloned EBV genome in mammalian cells with biological activity and stability. The goal in this aim will be to recover infectious virus after introductionof assembled herpesvirus genomes into mammalian cells. EBV assembled genomes can be transfected into HEK-293 or EBV negative Akata cells. Cells that harbor the EBV episome, following drug selection, will be induced for lytic virus production. Biological activity will be measured using quantitative PCR for viral genomes, Raji GFP titers that measure establishment of latency and reactivation and finally by the ability to immortalize B cells. Our singular goal isto use the combined and complementary expertise of the JHU and JCVI laboratories to demonstrate we can assemble whole genome infectious clones of herpesviruses from the individual parts in an efficient process with high fidelity and stability. If successful, this woul provide a new powerful platform to clone and manipulate these viruses.

 描述（由申请方提供）：疱疹病毒是大DNA病毒，其基因组具有超过100种基因产物的编码潜力。从历史上看，这些大基因组的遗传操作仅对这些病毒的一个子集是可行的。随后，许多疱疹病毒基因组被克隆到BAC质粒中，这显著地推进了在大肠杆菌宿主中的基因组工程技术和在适当的宿主细胞中成功地重建感染性病毒。在本申请中，我们 他们建议使用合成生物学首先构建EB病毒（EBV）基因组的野生型克隆，然后重建感染性病毒。将使用单纯疱疹病毒1型（HSV-1）作为模型，首先测试和优化组装方法。这种合成生物学方法的成功结果将对合成克隆和操纵任何疱疹病毒基因组的能力产生变革性影响。此外，这种方法提供了一个范例，以帮助了解新兴病原体的分子遗传学和生物学。具体目标1。使用合成基因组学方法组装EBV的感染性基因组。在这个目标中，我们将克隆EBV基因组，株Akata，使用合成基因组学。我们的方法将基于J.克雷格文特尔研究所（JCVI）团队成员在该领域的最新进展，该团队创造了第一个合成微生物。JCVI实验室已经开发出能够组装大DNA片段的方法，从数百个酶到百万倍大小的基因组。该团队将使用这些方法组装EBV的感染性克隆，同时使用更“易处理”的HSV-1基因组组装来优化和改进合成基因组学方法。具体目标2。在哺乳动物细胞中建立具有生物学活性和稳定性的克隆EBV基因组。这一目标的目标将是在将组装好的疱疹病毒基因组导入哺乳动物细胞后回收感染性病毒。EBV组装的基因组可以转染到HEK-293或EBV阴性Akata细胞中。在药物选择后，将诱导携带EBV附加体的细胞产生裂解性病毒。将使用病毒基因组的定量PCR、测量潜伏期和再活化的建立的Raji GFP滴度以及最终通过使B细胞永生化的能力来测量生物活性。我们的唯一目标是利用JHU和JCVI实验室的联合和互补专业知识，证明我们可以在一个高效的过程中，以高保真度和稳定性，从单个部分组装疱疹病毒的全基因组感染性克隆。如果成功，这将为克隆和操纵这些病毒提供一个新的强大平台。