NUCLEOCYTOPLASM EXPORT OF SIMIAN RETROVIRUS RNA GENET ELEMENT & PROTEIN FAC:AIDS

猿逆转录病毒RNA基因元件的核质输出

• 批准号: 6592301
• 负责人: CURTIS A MACHIDA
• 金额: $ 11.11万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6592301
• 关键词: AIDS; Mammalia; animal tissue; communicable diseases; immunology; inflammation; lymphatic system; nucleic acids; proteins; virus

The simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem-loop structure containing two major internal loops exhibiting 180? inverse symmetry, and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region=s known stable stem-loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3' IR or sub-region RNAs, and that formation of RNA-protein complexes are conferred by interaction with other novel proteins. UV-crosslinking of RNA-protei n complexes, coupled with RNase T1 digestion and denaturing polyacrylamide gel electrophoresis, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual sub-region RNAs, apparently recognizing the CTE RNA at multiple sites of interaction. In addition, we constructed a series of SRV infectious clone recombinants containing varying permutations of sub-region fragments. RNA slot-blot analyses indicate that recombinants containing the complete CTE in sense orientation, or containing two contiguous sub-regions that reconstitute the 3' two-thirds of this region, can facilitate or partially facilitate SRV RNA export in transfectant cells. These experiments indicate that sequence determinants of the 3' IR, in addition to secondary structure, are important factors in the nuclear export of unspliced SRV RNA, and that proteins other than RNA helicase A may also directly recognize the serogroup 2 SRV CTE. FUNDING NIH DK53462 PUBLICATIONS Li B, Wyman T, Moudgil T, Marracci G, Ju CF, Machida CA. Nuclear export of simian retroviral RNA Critical genetic elements and cellular protein factors. In Program of the Sixteenth Annual Symposium on Nonhuman Primate Models for AIDS (held in Atlanta, GA, October 7-10, 1998) (abstract 79).

猴反转录病毒(SRV)基因组包含一个构成成分 其3‘间隔区(IR)内的运输元件(CTE) 介导未剪接SRV RNA的核输出。血清组2 SRV CTE预计会形成一个稳定的茎环结构，其中包含两个 主要内部环路显示为180？逆对称，以及附加 较小的内部和终端环路。开始对…进行鉴定 潜在的CTE相互作用蛋白和评估结构 对于蛋白质相互作用的要求，我们进行了RNA迁移率的移动 使用消除该区域的IR片段的分析=S已知稳定 茎-环结构。使用免疫印迹分析，我们已经确定 与未剪接SRV核出口有关的RNA解旋酶A 基因组RNA，似乎并不直接与 完整的血清组2 SRV 3‘IR或亚区RNA，以及该形成 RNA-蛋白质复合体是通过与其他新的 蛋白质。RNA-蛋白质复合体的紫外光交联 核糖核酸酶T1消化和变性聚丙烯酰胺凝胶电泳法， 表明一种新的120 kDa的蛋白质相互作用 使用完整的CTE或单独的亚区RNA，显然 在多个相互作用位点识别CTE RNA。在……里面 此外，我们还构建了一系列SRV感染性克隆重组体 包含不同排列的亚区片段。核糖核酸 狭缝杂交分析表明，含有完整基因的重组体 有义方向的CTE，或包含两个相邻子区域的CTE 重新构成了这个地区的3‘2/3，可以促进或 部分促进SRV RNA在转基因细胞中的输出。这些 实验表明，3‘IR，In的序列决定因素 除了二级结构，都是核中的重要因素 输出未剪接SRV RNA和RNA解旋酶以外的蛋白质 A也可以直接识别血清组2 SRV CTE。资助美国国立卫生研究院 DK53462出版物李斌，怀曼T，Moudgil T，Marracci G，Ju CF 加利福尼亚州马奇达。猴反转录病毒关键基因的核输出 元素和细胞蛋白因子。在十六大规划中 艾滋病非人类灵长类模型年度研讨会(在亚特兰大举行， 1998年10月7日至10日)(摘要79)。