Engineering Herpesviruses using Synthetic Genomics

 描述(申请人提供)：疱疹病毒是一种大型DNA病毒，其基因组具有超过100个基因产物的编码潜力。从历史上看，对这些大基因组的遗传操作只适用于这些病毒的一部分。随后，许多疱疹病毒基因组被克隆到BAC载体中，极大地促进了大肠杆菌宿主的基因组工程技术和感染性病毒在合适的宿主细胞中的成功重组。在此应用程序中，我们 建议利用合成生物学首先构建EB病毒(EBV)基因组的野生型克隆，然后重构传染性病毒。将以单纯疱疹病毒1型(HSV-1)为模型，首先对组装方法进行测试和优化。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革命性影响。此外，这种方法提供了一种范式来帮助理解新出现的病原体的分子遗传学和生物学。具体目的1.利用合成基因组学方法组装EBV感染性基因组。在这个目标中，我们将使用合成基因组学来克隆EBV基因组Akata株。我们的方法将基于J·克雷格·文特尔研究所(JCVI)团队成员在这一领域取得的最新进展，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片段DNA的方法，大小从百千碱基到百万碱基大小的基因组。该团队将使用这些方法来组装具有感染性的EBV克隆，同时使用更容易处理的HSV-1基因组组装来优化和提炼合成基因组学方法。具体目的2.在哺乳动物细胞中建立具有生物学活性和稳定性的EBV克隆基因组。这一目标的目标将是在将组装的疱疹病毒基因组导入哺乳动物细胞后恢复传染性病毒。EBV组装的基因组可导入HEK-293或EBV阴性的AKATA细胞。携带EB病毒Episome的细胞，在药物选择后，将被诱导产生裂解病毒。病毒基因组的生物活性将通过定量聚合酶链式反应来测量，Raji GFP滴度将测量潜伏期和重新激活的建立，最后通过使B细胞永生的能力来测量。我们的唯一目标是利用JHU和JCVI实验室结合和互补的专业知识来证明我们可以以高保真和稳定的高效过程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，这将提供一个新的强大的平台来克隆和操纵这些病毒。

 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.