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: 6453677
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6453677
• 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).

猴逆转录病毒（SRV）基因组含有一个组成型 在其3'基因间区（IR）内的转运元件（CTE）， 介导未剪接的SRV RNA的核输出。 血清型2型SRV CTE被预测为形成稳定的茎环结构，其包含两个 主要的内部循环表现出180？ 逆对称，以及附加的 次要的内部和终端环路。 开始识别 潜在的CTE相互作用蛋白，并评估结构 为了满足蛋白质相互作用的需要，我们进行了RNA迁移率改变， 使用消除该区域的IR片段的测定=已知稳定的 茎环结构 使用免疫印迹分析，我们已经确定 RNA解旋酶A，与未剪接的SRV的核输出有关， 基因组RNA，似乎并不直接与 完整的血清组2SRV 3 ′ IR或亚区RNA， 的RNA-蛋白质复合物是通过与其他新的 proteins. RNA-蛋白质复合物的UV交联，与 RNase T1消化和变性聚丙烯酰胺凝胶电泳， 表明一种分子量为120 kDa的新型蛋白质与 与完整CTE或单个亚区域RNA，显然 在多个相互作用位点识别CTE RNA。 在 此外，我们还构建了一系列的SRV感染性克隆重组子 包含子区域片段的不同排列。 RNA 狭缝印迹分析表明，含有完整的 有义方向的CTE，或包含两个连续的子区域 重新构成了这个地区的三分之二，可以促进或 部分促进转染细胞中的SRV RNA输出。 这些 实验表明，序列决定簇的3' IR，在 除了二级结构，是重要的因素，在核 未剪接的SRV RNA的输出，以及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NIH Li B，Wyman T，Moudgil T，Marracci G，Ju CF， 町田CA. 猴逆转录病毒RNA的核输出 元素和细胞蛋白因子。 在第十六届的节目中 艾滋病非人灵长类模型年度研讨会（在亚特兰大举行， GA，1998年10月7日至10日）（摘要79）。