Vaccinia Virus Genetics and Morphogenesis

描述（由申请人提供）：本提案的目标是完成来自两个不同突变体，Dales和Condit的132个温度敏感痘苗病毒突变体的物理定位和表型分析，并详细研究这些突变体的定义子集，以完善我们对病毒粒子形态发生的理解。在Aim 1中，利用标记拯救技术，将牛痘病毒温度敏感突变体的des - condit组合集合中每个未定位或部分定位的互补组的突变体定位到单个基因上。在第2项研究中，迄今为止，从戴尔-康迪特联合收集的每个互补组中未表征的代表将进行一级表型分析，包括病毒DNA复制、病毒蛋白合成和病毒粒子形态发生的测定。在目标3中，从合并的Dales- Condit集合中选择互补组的代表将对病毒粒子形态发生缺陷进行详细分析。鉴定将包括病毒粒子温度敏感性测定、病毒蛋白翻译后切割、DNA包装、合成、病毒粒子定位和受影响基因蛋白产物的细胞内定位、在非许可条件下感染细胞的详细电镜分析以及累积的异常中间颗粒的生化分析。该项目的成功完成将增强我们对痘病毒基因组功能组织的理解，为今后痘病毒功能基因组学的研究提供广泛的永久痘苗病毒条件致死性突变体的收集，并阐明病毒形态发生的遗传和生化途径。从历史上看，病毒形态发生的研究对我们对自组装系统、膜生物学、细胞内蛋白质运输和病毒细胞相互作用的理解产生了重大影响。我们完全期待我们的研究将产生类似的影响。重要的是，鉴于天花有可能被用作生物恐怖主义武器，这类基础天花病毒学研究对公共卫生的价值最近得到了显著提高。

DESCRIPTION (provided by applicant): The goals of this proposal are to complete the physical mapping and phenotypic analysis of a combined set of 132 temperature sensitive vaccinia virus mutants from two different mutant collections, Dales and Condit, and to study in detail a defined subset of these mutants in order to refine our understanding of virion morphogenesis. In Aim 1, mutants from each of the unmapped or partially mapped complementation groups in the combined Dales-Condit collection of vaccinia virus temperature sensitive mutants will be mapped to a single gene using marker rescue. In Aim 2, heretofore uncharacterized representatives from each complementation group in the combined Dales-Condit collection will be subjected to a first level phenotypic analysis, consisting of assays for viral DNA replication, viral protein synthesis, and virion morphogenesis. In Aim 3, representatives from selected complementation groups in the combined Dales- Condit collection will be subjected to detailed analysis for defects in virion morphogenesis. Characterization will include assays for virion temperature sensitivity, post-translational cleavage of viron proteins, DNA packaging, synthesis, virion localization and intracellular localization of the protein product of the affected gene, detailed electron microscopic analysis of cells infected under non-permissive conditions, and biochemical analysis of accumulated aberrant intermediate particles. The successful completion of this project will enhance our understanding of the functional organization of the poxvirus genome, it will provide for the scientific community an extensive permanent collection of vaccinia virus conditional lethal mutants for future use in poxvirus functional genomics, and it will elucidate a genetic and biochemical pathway for virus morphogenesis. Historically, studies of virus morphogenesis have impacted significantly on our understanding of self assembly systems, membrane biology, intracellular protein trafficking, and virus cell interactions. We fully expect that our studies will have a similar impact. Importantly, the value of this type of research in basic poxvirology to public health has been significantly increased recently given the potential for use of smallpox as a bioterrorist weapon.