REGULATORY ELEMENTS OF AVIAN SARCOMA VIRUS LTRS

禽肉瘤病毒 LTRS 的调控元件

• 批准号: 2095730
• 负责人: RAMAREDDY V GUNTAKA
• 金额: $ 13.09万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2095730
• 关键词: RNase protection assay; Rous sarcoma virus; antibody; chemical binding; chickens; complementary DNA; fibroblasts; gel mobility shift assay; gene deletion mutation; gene expression; genetic regulation; genetic regulatory element; genome; host organism interaction; molecular cloning; nucleic acid repetitive sequence; phosphorylation; point mutation; polymerase chain reaction; posttranscriptional RNA processing; transcription factor; transfection; virus genetics

The overall objective of this research project is to understand the host transcription factors involved in the determination of species-specific and tissue-specific expression of Rous sarcoma virus genome. The long terminal repeats (LTR) of the virus contain powerful cis-acting elements to which host factors bind and activate transcription. For some time we have been studying in detail the molecular reactions between host factors and cis- sequences in the promoter-enhancer region (U3) of the RSV lTR. We, and others, have found that at least three well-defined cis elements are required for maximal expression of LTR-linked genes and three different factors interact with these motifs. We have also succeeded in isolating three different yet related cDNA clones encoding polypeptides that specifically bind to distinct sequence motifs. We have shown that one of these factors, mainly present in the avian fibroblasts, and in muscle tissue, binds to a specific site, E3/E3-1, localized at nucleotides -151 to -171. The importance of this cis-acting motif for viral LTR function has not ben examined. The aims of the current study are; (i) to define the role of the cis-acting sequence E3/E3-1 (-151 to -171) in LTR function and characterize the protein binding to this sequence as it appears that this factor is present only in avian fibroblasts, the target tissue for efficient expression of viral genome; (ii) to determine the functional domains of the cloned factors and their cognate cis-acting sequences; (iii) to establish the role of the cloned cDNA products in RSV gene expression. In-frame deletions and point mutations will be made in the cDNAs to identify the various DNA binding domains of the factors. We will also address the issue of whether phosphorylation and homo- or heterodimerization of various factors are required for their interaction, using antibodies raised against the purified factors. These studies will provide further insights into the regulation of viral gene expression and the contribution of host factors in determining tissue tropism and species- specific expression of viral genes. Preliminary results indicated that the GT-rich sequence of the U5 region of the lTR contributes to transcription termination and/or the 3' end processing of the RNA. We intend to continue these studies with an objective of identifying precisely the nucleotides involved in the 3' end maturation of the mRNA, which will be determined by S1 protection assays, oligonucleotide-directed mutagenesis and by studies using polymerase chain reaction.

本研究项目的总体目标是了解东道主 转录因子参与确定物种特异性和 Rous肉瘤病毒基因组的组织特异性表达。长长的终端 病毒的重复序列(LTR)包含强大的顺式作用元件，对其 宿主因子结合并激活转录。一段时间以来，我们一直是 详细研究了寄主因子与顺式分子间的分子反应。 RSV ltr启动子-增强子区域(U3)的序列。我们，还有 其他人则发现，至少有三个定义良好的顺式元素是 最大限度地表达LTR连锁基因所需的和三个不同的 因素与这些主题相互作用。我们还成功地分离出 三个不同但相关的编码多肽的cDNA克隆 与不同的序列基序特异性结合。我们已经证明了其中一个 这些因子主要存在于禽类成纤维细胞和肌肉中 组织，结合到特定的位点，E3/E3-1，定位于核苷酸-151到 -171.这个顺式作用基序对病毒LTR功能的重要性 不是本检查过的。这项研究的目的是：(I)界定 顺式作用序列E3/E3-1(-151至-171)在LTR功能中的作用 描述与这个序列结合的蛋白质，因为这似乎是 因子只存在于禽类成纤维细胞中，其靶组织是 病毒基因组的高效表达；(Ii)确定功能 克隆因子及其同源顺式作用序列的结构域； 目的：探讨克隆产物在呼吸道合胞病毒基因表达中的作用。 框内缺失和点突变将在cDNA中进行 鉴定这些因子的各种DNA结合域。我们还将 解决的问题是磷酸化和同型或 它们的相互作用需要各种因素的异二聚化， 使用针对纯化因子产生的抗体。这些研究将 提供对病毒基因表达调控的进一步见解和 寄主因素在决定组织嗜性和物种中的作用 病毒基因的特异性表达。初步结果显示， LTRU5区富含GT的序列有助于转录 RNA的终止和/或3‘端处理。我们打算继续 这些研究的目的是准确地识别核苷酸 参与信使核糖核酸的3‘端成熟，这将由 S1保护分析、寡核苷酸导向的突变和研究 使用聚合酶链式反应。