GENE REGULATION OF MAMMALIAN DNA VIRUSES

我们将重点介绍痘病毒分子生物学的三个方面：(1) 这些病毒选择性动员的机制 胞质中的核成分以及蛋白质是否 (2)晚期病毒基因的酶学研究 宿主细胞RNA转录及其在这一过程中的作用 聚合酶II(POL II)；以及(3)鉴定和消除 与鸡传染性支气管炎多种致病特性相关的病毒基因 病毒。痘病毒感染对α-Amanitin和 病毒未能在去核细胞中生长表明 细胞外Pol II是病毒生产所必需的。我们已经展示了 痘病毒诱导POL最大亚基易位 II进入细胞质。痘病毒致病机制的研究 招募核组件需要进行能力测试 病毒成分来诱导易位。我们希望实现 这是通过：(1)显微注射入青蛙卵母细胞或哺乳动物细胞； (2)与酵母球形体融合；或(3)添加病毒 组分直接进入孤立的原子核。为了调查 酶学和POL II在病毒晚期基因转录中的作用 我们将继续制备或获得抗POL的单抗 II和病毒RNA聚合酶。抗体，再加上 酶分析将使我们能够跟踪病毒和宿主的命运 感染后期的RNA聚合酶亚基 了解Late的组成和功能 转录复合体。最终，我们希望净化一个复合体 能够在体外识别晚期启动子。另一个主要目标 是研究痘病毒致病的分子基础。 痘病毒相关基因的鉴定 由于对重组人的兴趣，致病机制是及时的 痘苗病毒作为疫苗载体。我们描述了一种无毒的 病毒删除突变体，仅在 并不能在中枢或内脏内生长 关于动物的。我们将改造这个突变体，使其包含一个独特的位点 用于重新插入已删除的区域，从而识别 允许病毒在中枢神经系统和内脏内生长的基因。 我们将尝试将发病增加与疾病的变化联系起来 宿主在文化中的范围，可以作为一个简单的索引 潜在的疫苗并发症和副作用。

We will focus on three areas of poxvirus molecular biology: (1) the mechanism by which these viruses selectively mobilize nuclear components to the cytoplasm and whether proteins to be mobilized are modified; (2) the enzymology of late viral gene transcription and the role in this process of host cell RNA polymerase II (Pol II); and (3) the identification and elimination of viral genes associated with various pathogenic properties of the virus. The sensitivity of poxvirus infections to alpha-amanitin and the failure of the virus to grow in enucleated cells suggest that cellular Pol II is required for virus production. We have shown that poxviruses induce translocation of the largest subunit of Pol II to the cytoplasm. A study of the mechanism by which poxvirus recruits nuclear components necessitates an assay for the ability of viral components to induce translocation. We hope to achieve this by: (1) microinjection into frog oocytes or mammalian cells; (2) fusion with yeast spheroplasts; or (3) the addition of viral components directly to isolated nuclei. To investigate the enzymology and the role of Pol II in late viral gene transcription, we will continue to prepare or obtain monoclonal antibodies to Pol II and the viral RNA polymerase. The antibodies, coupled with enzyme assays will allow us to follow the fates of viral and host RNA polymerase subunits during the later phases of infection to gain insight about the composition and function of the late transcription complex. Ultimately, we wish to purify a complex capable of late promoter recognition in vitro. Another major goal is to study the molecular basis of pathogenesis of poxviruses. Identification of genes associated with aspects of poxvirus pathogenesis is timely because of the interest in recombinant vaccinia viruses as vaccine vectors. We describe an avirulent virus deletion mutant which grows only locally during scarification and is incapable of growth within the CNS or viscera of animals. We will engineer this mutant to contain a unique site for reinsertion of the deleted regions and thereby identify the genes which permit the virus to grow within the CNS and viscera. We will try to correlate increases in pathogenesis with changes in host range in culture which might serve as a simple index of potential vaccine complications and side effects.