VACCINIA VIRUS BIOCHEMICAL GENETICS

本项目的长期目标是了解 牛痘病毒基因组的功能组织， 调节牛痘病毒基因表达的机制， 以及牛痘病毒与宿主细胞之间的相互作用。 的 为实现这些目标而采取的方法是隔离和 病毒条件致死突变体的功能分析。 具体地，本申请提出详细表征 先前分离的温度敏感突变似乎 影响牛痘生物学的三个明显不同的方面。 病毒转录将通过研究几个 影响两种病毒RNA的温度敏感性突变 聚合酶亚单位。 RNA的转录后调控 稳定性将通过研究温度敏感性、药物 耐药和药物依赖性突变体都与“流产”有关， 晚期”突变体表型。 将通过以下方法研究病毒体形态发生： 分析了大量的温度敏感突变体， 在非感染部位赋予“正常”表型 允许温度 本申请还建议继续开发 牛痘基因的体外定点诱变方法。 使用最近开发的用于有效插入 将未选择的标记插入牛痘基因组中，尝试将是 构建温度敏感突变， 在化学诱变确定的病毒DNA后， 体外 实验也将进行测试的有用性 一个新构建的无义抑制细胞系， 分离牛痘病毒无义突变体。

The long term goals of this project are to understand the functional organization of the vaccinia virus genome, the mechanisms by which vaccinia virus gene expression is regulated, and the interactions between vaccinia virus and the host cell. The approach taken to achieve these goals is the isolation and functional analysis of conditionally lethal mutants of the virus. Specifically, this application proposes to characterize in detail previously isolated temperature sensitive mutations which seem to affect three distinctly different aspects of vaccinia biology. Viral transcription will be addressed by studying several temperature sensitive mutations affecting two of the viral RNA polymerase subunits. Post transcriptional regulation of RNA stability will be addressed by studying temperature sensitive, drug resistant, and drug dependent mutants all related to an "abortive late" mutant phenotype. Virion morphogenesis will be studied by analyzing a large group of temperature sensitive mutants which confer a "normal" phenotype on an infection done at the non- permissive temperature. This application also proposes to continue the development of methods for site directed in vitro mutagenesis of vaccinia genes. Using a recently developed protocol for efficient insertion of unselected markers into the vaccinia genome, attempts will be made to construct temperature sensitive mutations in specific viral genes after chemical mutagenesis of defined viral DNA's in vitro. Experiments will also be performed to test the usefulness of a newly constructed nonsense suppressing cell line for the isolation of nonsense mutants of vaccinia virus.