GENE REGULATION OF MAMMALIAN DNA VIRUSES

我们将集中在痘病毒分子生物学的三个领域：（1） 这些病毒选择性移动的机制 核成分到细胞质，以及蛋白质是否被 （2）晚期病毒基因的酶学 转录和宿主细胞RNA在这一过程中的作用 聚合酶II（Pol II）;和（3）鉴定和消除 与各种致病特性相关的病毒基因 病毒 痘病毒感染对α-鹅膏蕈碱和 病毒不能在去核细胞中生长表明， 细胞Pol II是病毒生产所必需的。 我们已经表明 痘病毒诱导Pol最大亚基易位， II到细胞质。 痘病毒感染机体机制的研究 招募的核组件需要测试 病毒成分来诱导易位。 我们希望实现 （1）显微注射入蛙卵母细胞或哺乳动物细胞; (2)与酵母原生质球融合;或（3）加入病毒 成分直接分离到细胞核。 探讨 酶学和Pol II在晚期病毒基因转录中的作用， 我们将继续制备或获得抗Pol的单克隆抗体， II和病毒RNA聚合酶。 抗体，加上 酶分析将使我们能够跟踪病毒和宿主的命运， RNA聚合酶亚基在感染后期， 深入了解后期的组成和功能 转录复合体 最终，我们希望净化一个复杂的 能够在体外识别晚期启动子。 另一个主要目标 研究痘病毒致病的分子基础。 痘病毒相关基因的鉴定 发病机制是及时的，因为对重组 牛痘病毒作为疫苗载体。 我们描述了一种无毒的 病毒缺失突变体，仅在局部生长 划痕，不能在CNS或内脏内生长 动物。 我们会改造这个突变体， 用于重新插入缺失的区域，从而鉴定 允许病毒在中枢神经系统和内脏内生长的基因。 我们将尝试将发病机制的增加与 在文化的寄主范围，这可能作为一个简单的指标， 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.