Synthetic Genomics Approach to Assemble Infectious Clones of KSHV

组装 KSHV 感染性克隆的合成基因组学方法

• 批准号: 9807969
• 负责人: PRASHANT J DESAI
• 金额: $ 10.56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9807969
• 关键词: Antigens; B-Lymphocytes; Bacterial Artificial Chromosomes; Basic Science; Biological; Biology; Cell Line; Cells; Chemicals; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complement; Complex; DNA; DNA Viruses; Endothelial Cells; Engineering; Episome; Escherichia coli; Generations; Genes; Genetic Recombination; Genome; Genome engineering; Genomic approach; Goals; Herpesviridae; Herpesvirus 1; Human Herpesvirus 4; Human Herpesvirus 8; In Vitro; Individual; Institutes; Laboratories; Lytic; Lytic Virus; Mammalian Cell; Measures; Methods; Microbe; Monitor; Outcome; Pharmaceutical Preparations; Plasmids; Procedures; Process; Production; Property; Reagent; Reporting; Scientist; Stains; Structure; Tars; Technology; Telomerase; Time; Titrations; Translating; Viral Genome; Virus; Yeasts; base; established cell line; gene product; genetic analysis; genetic manipulation; homologous recombination; latent gene expression; lytic gene expression; member; mutant; reconstitution; reverse genetics; synthetic biology; synthetic genomics; tool; virus culture

The DNA genomes of herpesviruses range in size from 120 kb to 240 kb and hence, have a large coding capacity in some cases in excess of 100 gene products. For years, genetic manipulation of these 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 a transformational approach, which is to use synthetic biology to build wild-type clones of the Kaposi's sarcoma- associated herpesvirus (KSHV) genome and demonstrate the reconstitution of infectivity of these assembled genomes. The successful outcome of this synthetic genomics approach will significantly advance the ability to clone, assemble and engineer this important virus in a more high-throughput manner. Specific Aim 1: Use synthetic genomics methods to clone and assemble KSHV genomes in yeast. In this aim, we will clone the KSHV genomes from two strains, BCBL-1 and JSC-1, using synthetic genomics methods. These genomes will be deconstructed into 11 parts, which can be modified separate of each other and then reassembled using yeast homologous recombination. Our approach will be based on advances made in this field by members of the J. Craig Venter Institute (JCVI) team that created the first synthetic microbe. In a collaborative effort, we have already assembled an infectious clone of herpes simplex virus type-1 (HSV-1) using these methods and are close to completion of an Epstein-Barr virus (EBV) Akata genome. We will use these new and powerful synthetic genomics methods to assemble complete genomes of KSHV in yeast. Specific Aim 2: Reconstitute biological activity of the assembled KSHV genomes in mammalian cells. The goal in this aim will be to recover infectious virus after introduction of assembled herpesvirus genomes into mammalian cells. KSHV assembled genomes will be transfected/electroporated endothelial cells (TIME - telomerase-immortalized microvascular endothelial cells) and BJAB cells. Cell lines that harbor the KSHV episome, following drug selection, will be induced for lytic virus production. Biological activity will be measured using latency antigen (LANA) staining to measure establishment of latency as well as spindle cell conversion of endothelial cells. Virus reactivation following lytic activation will be determined using quantitative PCR to measure viral genomes, lytic gene expression as well as TIME GFP titers. Our singular goal is to use the combined and complementary expertise of the JHU and JCVI laboratories to demonstrate we can assemble complete genomes of herpesviruses from the individual parts in an efficient process with high fidelity and stability. If successful, this would provide a new powerful platform to clone and manipulate these viruses to facilitate the study of their biology. This technology will thus, complement and extend the existing BAC methods.

疱疹病毒的DNA基因组的大小范围为120 kb至240 kb，因此具有大的编码区。 在某些情况下超过100个基因产物的能力。多年来，对这些基因组的遗传操作 仅对这些病毒的一部分是可行的。随后，许多疱疹病毒基因组被克隆 转化为BAC质粒，这大大推进了大肠杆菌宿主中的基因组工程技术， 感染性病毒在适当的宿主细胞中成功重建。在本申请中，我们提出了一种 转化方法，即利用合成生物学构建卡波西肉瘤的野生型克隆， 相关疱疹病毒（KSHV）基因组，并证明这些组装的感染性重建 基因组这种合成基因组学方法的成功结果将大大提高 克隆、组装和以更高通量的方式设计这种重要的病毒。 具体目标1：使用合成基因组学方法在酵母中克隆和组装KSHV基因组。在 为此，我们将利用合成基因组学技术，从两株KSHV，BCBL-1和JSC-1中克隆基因组 方法.这些基因组将被分解为11个部分，每个部分可以单独修改 然后使用酵母同源重组进行重组。我们的方法将基于取得的进展 在这一领域，由J.克雷格文特尔研究所（JCVI）的成员创造了第一个合成微生物。中 通过共同努力，我们已经组装了1型单纯疱疹病毒（HSV-1）的感染性克隆 使用这些方法，并接近完成Epstein-Barr病毒（EBV）Akata基因组。我们将使用 这些新的和强大的合成基因组学方法组装KSHV在酵母中的完整基因组。 具体目标2：在哺乳动物细胞中重建组装的KSHV基因组的生物活性。 该目的的目标将是在将组装的疱疹病毒基因组引入宿主细胞后回收感染性病毒。 哺乳动物细胞KSHV组装的基因组将被转染/电穿孔的内皮细胞（TIME -2010）。 端粒酶永生化微血管内皮细胞）和BJAB细胞。携带KSHV的细胞系 药物选择后，附加体将被诱导产生裂解病毒。将测量生物活性 使用潜伏抗原（拉娜）染色来测量潜伏期的建立以及梭形细胞转化， 内皮细胞将使用定量PCR测定裂解活化后的病毒再活化， 测量病毒基因组、裂解基因表达以及TIME GFP滴度。 我们的唯一目标是利用JHU和JCVI实验室的综合和互补专业知识， 证明我们可以从单个部分组装完整的疱疹病毒基因组， 具有高保真度和稳定性。如果成功，这将提供一个新的强大平台， 操纵这些病毒以促进其生物学的研究。这项技术将补充和 扩展了现有的BAC方法。