RETROVIRUS REGULATION IN VITRO AND DURING DEVELOPMENT

拟议实验的长期目标是了解如何 逆转录病毒序列介导在允许条件下基因表达控制 条件，例如在引入培养细胞的质粒分子中 以及这些序列如何克服发育信号， 染色质结构变化和基因表达异常 插入基因组后位于细胞DNA侧翼。 这些问题 对于理解病毒调节和病毒 发病机制，特别是整合的前病毒的能力， 调节原病毒中邻近细胞癌基因的表达， 诱导的肿瘤衍生细胞。 以前的工作主要集中在禽内源性病毒的表达 以及整合前病毒之间的相互作用 和邻近的细胞DNA 本提案的重点是病毒 在禽类红细胞生成过程中的表达以及 整合前病毒对chormatin结构和转录的影响 这些细胞中侧翼宿主序列的活性。 这将是 使用内源性原病毒ev-6完成，其被驱动离开内源性原病毒ev-6。 细胞启动子。 分析该病毒和邻近宿主的序列 因此可以研究前病毒的作用 整合对已知启动子元件的活性的影响。 先前 结果表明，分化红细胞是一个有趣的过程， 系统来研究序列内容之间的关系， 染色质结构和转录，因为事实上，虽然这些 细胞经历一个整体的浓缩和转录失活， 大多数细胞基因，前病毒DNA能够逃脱这两个 流程. 第二组实验旨在补充和扩展体内 上述实验通过直接分析三个 来自致病性、外源性病毒和非致病性的病毒序列， 内源性病毒在相连LTR的转录调节中的作用。 的 这些研究的目的是确定参与转录的序列 病毒和邻近宿主特异性序列的调节。 三 将检测的内源性和外源性病毒区域为 来自前病毒LTR的U3区，env基因中的一个区域， 与其他前病毒和细胞转录调节区， 包括在核酸酶超敏感位点中，并被特异性保护， 从发育过程中的从头甲基化和3'非翻译 来自内源性病毒的区域，该区域涉及抑制 连接的LTR的增强子/启动子活性。 这些实验将 通过分析重组的、转染的 质粒通过瞬时表达测定。

The long range goal of the proposed experiments is to understand how retroviral sequences mediate control of gene expression under permissive conditions such as in plasmid molecules introduced into cultured cells and how such sequences overcome developmental signals to induce chromatin structural changes and aberrant expression of genes in flanking cellular DNA after insertion into the genome. These questions are important in understanding both viral regulation and viral pathogenesis, particularly the ability of integrated proviruses to modulate the expression of adjacent cellular oncogenes in provirus- induced tumor-derived cells. Previous work has focused on the expression of avain endogenous viruses during development, and the interactions between integrated proviruses and adjacent cellular DNA. The focus in this proposal is on virus expression during avian erythorpoiesis and the cis-effects induced by integrated proviruses on the chormatin structure and transcriptional activity of flanking host sequences in these cells. This will be accomplished using the endogenous provirus ev-6, which is drived off of a cellular promoter. Analysis of this virus and adjacent host sequences will therefore allow the investigation of the effects of provirus integration on the activity of a known promoter element. Previous results indicate that differentiating erythrocytes are an interesting system to investigate the relationship between sequence content, chromatin structure and transcription due to the fact that, while these cells undergo a global condensation and transcriptional inactivation of most cellular genes, proviral DNA is able to escape both of these processes. The second set of experiments seeks to complement and extend the in vivo experiments described above by directly analyzing the role of three viral sequences from pathogenic, exogenous viruses and non-pathogenic, endogenous viruses in transcriptional regulation of linked LTRs. The aim of these studies is to define sequences involved in transcription regulation of viral and adjacent host-specific sequences. The three regions from endogenous and exogenous viruses that will be tested are teh U3 regions from proviral LTRs, a region in the env gene which, as with other proviral and cellular transcriptional regulatory regions, is included in a nuclease hypersensitive site and is specifically protected from de novo methylation during development and the 3' untranslated region from endogenous viruses, which has been implicated in inhibiting the enhancer/promoter activity of linked LTRs. These experiments will be conducted by analyzing RNA expression of recombinant, transfected plasmids by transient expression assays.