Bacterial artificial chromosomes for HSV genomics

描述(申请人提供)：单纯疱疹病毒(HSV)是一种重要和常见的人类病原体。在美国，单纯疱疹病毒是导致非外伤性失明的主要原因，并伴有一系列眼部疾病，包括树突状上皮角膜炎、结膜炎和眼睑炎症，以及致盲的坏死性基质角膜炎。此外，单纯疱疹病毒会引起唇疱疹、生殖器溃疡，并且是病毒性脑炎的主要原因。在许多领域中，使用明确的基因改变来洞察基因的功能已经成为标准。这样的遗传方法通常是繁琐的，转基因生物的产生往往比实际的遗传功能分析本身耗费的时间要多得多。因此，有必要应用新技术来加快突变的产生。疱疹病毒的情况当然就是如此，尽管其庞大的DNA基因组可以通过同源重组来逆转遗传学，但它会使特定突变的产生复杂化。 这项提议的目标是利用细菌人工染色体(BAC)技术的力量，使HSV适应细菌遗传方法。对于其他一些疱疹病毒，BAC技术允许在不到一周的时间内产生几个突变。这与目前允许在2-3个月内产生单个突变体的HSV重组方法形成了鲜明对比。因此，将分别为三种主要的实验室HSV-1毒株和两种HSV-2毒株产生BAC。在将它们用作诱变模板之前，将从这些BAC中的每一个再生病毒，并将其表型与其原始亲本菌株进行仔细的比较。这将确保像BAC这样的病毒的传播不会固有地导致这些病毒的基因表达谱、毒力或致病机制发生任何未明确的变化。一旦建立并表征了这些试剂，这些试剂将被存放在ATCC，以便所有研究人员都能获得这项强大的技术。这项工作将代表着该领域的重大进展，即允许以标准化方式快速产生HSV突变，用于基础研究，以及疫苗、抗肿瘤药物和基因递送载体的开发。这项建议的成功结果与NEI角膜疾病计划的R03计划目标和战略目标一致，将对所有致力于HSV感染及其致盲后遗症的实验室的研究产生重大影响。

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) is a significant and common human pathogen. HSV is a leading cause of nontraumatic blindness in the US with an accompanying ocular diseases ranging from dendritic epithelial keratitis, conjunctivits and blepharitis, to blinding necrotizing stromal keratitis. In addition, HSV causes cold sores, genital sores, and is a leading cause of viral encephalitis. The use of defined genetic alterations has become standard in many fields to gain insight into the functions of genes. Such genetic approaches -are often cumbersome, with the generation of genetically altered organisms often being far more time-consuming than the actual analysis of genetic function itself. There is, therefore, a need for the application of novel technologies to speed up the generation of mutants. This is certainly the case for the herpes viruses whose large DNA genomes, although amenable to reverse genetics by homologous recombination, complicate the generation of defined mutants. The goal of this proposal is to harness the power of bacterial artificial chromosome (BAC) technology to make HSV amenable to bacterial genetic approaches. For some other herpes viruses, BAC technology allows the generation of several mutants in less than a week. This is in contrast to current HSV recombination methodologies that allow generation of a single mutant in 2-3 months. BACs will therefore be generated for each of the three major laboratory strains of HSV-1 and two strains of HSV-2. Prior to their use as templates for mutagenesis, viruses will be regenerated from each of these BACs and their phenotypes compared carefully to their original parental strains. This will ensure that the propagation of such viruses as BACs does not inherently cause any undefined changes in the gene expression profiles, virulence, or pathogenesis of any of these viruses. Once established and characterized these reagents will be deposited with ATCC to allow all researchers access to this powerful technology. This work will represent a major advance in the field in allowing the rapid generation of HSV mutants in a standardized fashion for basic research, as well as for vaccine, anti-tumor agent, and gene delivery vector development. The successful outcome of this proposal, consistent with the R03 program objectives and strategic goals of the NEI's Corneal Diseases Program, will have a major impact on the research of all laboratories working on HSV infections and their blinding sequelae.