MUTANTS OF EPSTEIN-BARR VIRUS

本提案中概述的实验的总体目标是 开发产生EBV突变病毒的方法。只有通过 这种突变体的构建和分析最终会变得清晰 哪些EBV基因是淋巴细胞增殖过程所必需的， 永生化、上皮细胞转化和肿瘤发生。的 实验依赖于三种不同的方法。在第一种方法中 天然存在的进化变体将被表征， 影响从潜伏期到病毒复制的进入途径， 淋巴细胞几个新的变异影响基因组的大小，配置， 和奥里利特的结构都在掌握之中。将详细研究这些问题。 第二种方法将尝试利用新创建的单元 具有病毒受体并且也允许病毒 复制的这些细胞将成为重组的基质， EBV毒株之间或一个毒株和另一个毒株的亚基因组DNA片段之间 完整的基因组第三种方法，我们有最初步的 数据，探索了重组基因盒的使用，其中可选择的 标记（用于耐药性）侧翼为EBV序列，目的是 完善EBV同源重组诱变的通用方法 其中EBV基因通过插入选择标记而失活。的 拟议中的实验是基于生物学和遗传学相结合的方法 这最终将增加我们对EBV基因和 基因产物相互作用产生如此多样的病理结果。

The overall objective of experiments outlined in this proposal is to develop methods for generating EBV mutant viruses. Only through construction and analysis of such mutants will it eventually become clear which EBV genes are essential for the process of lymphoid cell immortalization, epithelial cell transformation, and oncogenesis. The experiments rely on three different approaches. In the first approach naturally occurring evolutionary variants will be characterized which affect the entry pathway from latency into viral replication in lymphocytes. Several new variants which affect genome size, configuration, and the structure of orilyt are in hand. These will be studied in detail. The second approach will attempt to take advantage of newly created cell lines which have a virus receptor and are also permissive for virus replication. These cells will be the substrate for recombination between EBV strains or between subgenomic DNA fragments of one strain and another complete genome. The third approach, for which we have the most preliminary data, explores the use of recombinogenic cassettes in which a selectable marker (for drug resistance) is flanked by EBV sequences, The objective is to perfect a general method of EBV mutagenesis by homologous recombination in which EBV genes are inactivated by insertion of a selectable marker. The proposed experiments are based on combined biologic and genetic approaches which should eventually increase our understanding of how EBV genes and gene products interact to produce such diverse pathologic outcomes.