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).

猿猴逆转录病毒（SRV）基因组包含一个本构 在其3'3'间属区域内的运输元件（CTE）（ir） 介导未填充的SRV RNA的核出口。 血清2 SRV 预计CTE会形成一个稳定的茎环结构，其中包含两个 表现出180的主要内循环？ 对称和额外的对称性 次要内部和终端循环。 开始识别 潜在的CTE相互作用蛋白质并评估结构 蛋白质相互作用的要求，我们进行了RNA迁移率移动 使用IR片段的测定，该区域已知= S已知稳定 茎环结构。 使用免疫印迹测定，我们已经确定 RNA解旋酶A，与未填充SRV的核输出有关 基因组RNA，似乎并未直接与任何一个 完整的血清群2 srv 3'IR或子区域RNA，该形成 通过与其他新颖的相互作用赋予RNA-蛋白质复合物 蛋白质。 RNA蛋白质n络合物的紫外线连接，并与 RNase T1消化和变性聚丙烯酰胺凝胶电泳， 表明120 kDa分子量的新型蛋白质相互作用 使用完整的CTE或单个子区域RNA，显然 在多个相互作用位点识别CTE RNA。 在 此外，我们构建了一系列SRV感染性克隆重组 包含各个区域片段的不同排列。 RNA 插槽印刷分析表明，包含完整的重组者 CTE在有意义的方向上，或包含两个连续的子区域 重建该地区三分之二的三分之二可以促进或 部分促进了转染细胞中的SRV RNA输出。 这些 实验表明3'ir的序列决定因素， 增加二级结构是核的重要因素 导出未填充的SRV RNA，除RNA解旋酶以外的蛋白质 A也可以直接识别血清群2 SRV CTE。 资助NIH DK53462出版物Li B，Wyman T，Moudgil T，Marracci G，JU CF， 马克达 邻骨逆转录病毒RNA关键遗传的核输出 元素和细胞蛋白质因子。 在第16个计划中 艾滋病非人类灵长类动物模型的年度研讨会（在亚特兰大举行， GA，1998年10月7日至10日）（摘要79）。