Synthetic Genomics Approach to Assemble Infectious Clones of KSHV

疱疹病毒的dna基因组大小从120kb到240kb不等，因此具有很大的编码。 在某些情况下，生产能力超过100个基因产品。多年来，对这些基因组的遗传操作 对这些病毒中的一部分是可行的。随后，许多疱疹病毒基因组被克隆。 转化为BAC质粒，极大地促进了在大肠杆菌宿主和 在适当的宿主细胞中成功地重组了传染性病毒。在本申请中，我们提出了一种 变革性的方法，即使用合成生物学来构建卡波西肉瘤的野生型克隆- 相关疱疹病毒(KSHV)基因组和展示这些重组的传染性组装 基因组。这种合成基因组学方法的成功结果将极大地提高 以更高吞吐量的方式克隆、组装和设计这种重要的病毒。 具体目的1：利用合成基因组学方法在酵母中克隆和组装KSHV基因组。在……里面 为此，我们将利用合成基因组学方法克隆KSHV BCBL-1株和JSC-1株的基因组 方法：研究方法。这些基因组将被解构成11个部分，它们可以相互独立地修改 然后利用酵母同源重组进行重组。我们的方法将以取得的进展为基础 J·克雷格·文特尔研究所(JCVI)团队的成员在这一领域创造了第一个合成微生物。在一个 在合作的努力下，我们已经组装了一个具有传染性的1型单纯疱疹病毒(HSV-1)克隆 使用这些方法，并接近完成一个爱泼斯坦-巴尔病毒(EBV)赤田基因组。我们将使用 这些新的和强大的合成基因组学方法在酵母中组装KSHV的完整基因组。 特定目的2：在哺乳动物细胞中重建组装的KSHV基因组的生物学活性。 这个目标的目标是在将组装的疱疹病毒基因组引入到 哺乳动物细胞。KSHV组装的基因组将被转染/电穿孔内皮细胞(时间- 端粒酶永生化的微血管内皮细胞)和BJAB细胞。携带KSHV的细胞系 在药物选择之后，Episome将被诱导产生裂解病毒。生物活性将被测量 用潜伏期抗原(LANA)染色测量潜伏期的建立以及梭形细胞的转化 内皮细胞。裂解激活后的病毒重新激活将使用定量PCR来确定 测量病毒基因组、裂解基因表达以及时间GFP滴度。 我们的唯一目标是利用JHU和JCVI实验室的组合和互补的专业知识来 证明我们可以从单个部分高效地组装疱疹病毒的完整基因组 过程具有高保真度和稳定性。如果成功，这将提供一个新的强大的平台来克隆和 操纵这些病毒以便于研究它们的生物学。因此，这项技术将补充和 扩展现有的BAC方法。

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.