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（由申请人提供）： 疱疹病毒是大型 DNA 病毒，其基因组具有编码超过 100 个基因产物的潜力。历史上，这些大型基因组的基因操作仅适用于这些病毒的一个子集。基因组被克隆到 BAC 质粒中，这显着推进了大肠杆菌宿主中的基因组工程技术以及在适当的宿主细胞中成功重建感染性病毒的技术。 建议利用合成生物学首先构建 Epstein-Barr 病毒 (EBV) 基因组的野生型克隆，然后重建传染性病毒 1 型 (HSV-1) 将作为模型进行首次测试和研究。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作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来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 感染性基因组 在此目标中，我们将使用合成基因组学克隆 EBV 基因组 Akata 株。我们的方法将基于该领域成员的最新进展。 J. Craig Venter Institute (JCVI) 团队创造了第一个合成微生物 JCVI 实验室开发了能够组装从数百个碱基到兆碱基大小的基因组的方法。 EBV的感染性克隆，同时利用更“易处理”的HSV-1基因组组装来优化和完善合成基因组学方法。具体目标2.在哺乳动物细胞中建立具有生物活性和稳定性的克隆EBV基因组的目标。该目的是将组装的疱疹病毒基因组引入哺乳动物细胞中，然后将组装的基因组转染到含有 EBV 附加体的 HEK-293 或 EBV 阴性 Akata 细胞中，以恢复感染性病毒。将使用 PCR 来测量病毒基因组的生物活性，测量潜伏期和重新激活的建立，最后通过永生化 B 细胞的能力来测量。 JHU 和 JCVI 实验室的互补专业知识证明我们可以通过高保真度和稳定性的高效过程从各个部分组装疱疹病毒的全基因组感染性克隆。如果成功，这将提供一个新的强大平台。克隆和操纵这些病毒。