REGULATORY ELEMENTS OF AVIAN SARCOMA VIRUS LTRS

禽肉瘤病毒 LTRS 的调控元件

• 批准号: 2095729
• 负责人: RAMAREDDY V GUNTAKA
• 金额: $ 14.65万
• 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/2095729
• 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; virus virus interaction

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宿主 转录因子参与确定物种特异性和 劳斯肉瘤病毒基因组组织特异性表达。 的长末端 病毒的重复序列（LTR）含有强大的顺式作用元件， 宿主因子结合并激活转录。 一段时间以来， 详细研究了宿主因子与顺式- RSV ITR的启动子-增强子区（U3）中的序列。 我们也这么想 其他人已经发现，至少有三个明确的顺式元件， LTR连锁基因的最大表达所需的三个不同的 这些因素与这些图案相互作用。 我们还成功地分离出了 三种不同但相关的编码多肽的cDNA克隆， 特异性结合不同的序列基序。 我们已经证明， 这些因子主要存在于禽类成纤维细胞和肌肉中， 组织，结合到特定位点，E3/E3-1，位于核苷酸-151， -171. 这种顺式作用基序对病毒LTR功能的重要性已经被证实。 不是本检查的。 本研究的目的是：（一）确定 顺式作用序列E3/E3-1（-151至-171）在LTR功能中的作用， 表征与该序列结合的蛋白质，因为这似乎 因子仅存在于禽类成纤维细胞中， 病毒基因组的有效表达;（ii）确定功能性 克隆因子的结构域及其同源顺式作用序列;（iii） 以确定克隆的cDNA产物在RSV基因表达中的作用。 将在cDNA中进行框内缺失和点突变， 鉴定因子的各种DNA结合域。 我们还将 解决磷酸化和同源 各种因子的异二聚化是它们相互作用所必需的， 使用针对纯化因子的抗体。 这些研究将 为病毒基因表达的调控提供了进一步的见解， 宿主因素在决定组织趋向性和物种中的贡献- 病毒基因的特异性表达。 初步结果显示， lTR的U 5区的富含GT的序列有助于转录 终止和/或RNA的3'末端加工。 我们打算继续 这些研究的目的是精确地识别核苷酸， 参与mRNA的3'端成熟，这将通过以下方法确定： S1保护试验、阿昔替丁定向诱变和研究 使用聚合酶链式反应。