Post-Transcriptional Regulation of MMTV

MMTV 的转录后调控

基本信息

批准号：
7777297
负责人：
Jaquelin Page Dudley
金额：
$ 25.68万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7777297
关键词：
Address Affect Affinity Chromatography Avian Leukosis Betaretrovirus Binding Biological Biological Assay C-terminal Cell Differentiation process Cell Nucleus Cells Complement Complex Data Development Disease Elements Family member Gagging Genes Genetic Genome HERVs HIV Human Human T-Cell Leukemia Viruses In Vitro Introns Mammary gland Mediating Messenger RNA Modification Mouse Mammary Tumor Virus Murine leukemia virus Mus Mutation Names Nuclear Export Open Reading Frames Pathogenesis Pathway interactions Peptide Hydrolases Phosphorylation Post-Transcriptional Regulation Post-Translational Protein Processing Process Production Protein Export Pathway Proteins Proviruses RNA RNA Splicing RNA Stability RNA-Directed DNA Polymerase Recruitment Activity Reporter Retroviridae Specificity Structural Protein Superantigens Time Trans-Activators Transfection Translating Two-Hybrid System Techniques Viral Virus Replication Yeasts adapter protein base cell type dUTP pyrophosphatase genetic regulatory protein in vivo insight mRNA Export mammary tumor virus mouse model mutant novel research study rev Protein tissue culture vector viral RNA

项目摘要

Mouse mammary tumor virus (MMTV) has been classified as a simple retrovirus that encodes two accessory proteins, dUTPase (DU) and superantigen (Sag). The DU protein as well as Gag, protease (PR) and reverse transcriptase (RT) are encoded by unspliced viral RNA. Both simple and complex retroviruses require viral elements that facilitate the nuclear export of intron-containing mRNAs. Simple retroviruses have c/s-acting elements that directly recruit cellular factors involved in nuclear export, whereas complex retroviruses encode adapter proteins, such as Rev. Rev binds to viralc/s-acting sequences to facilitate cellular export factor recruitment. Our experiments indicate that MMTV encodes a third accessory protein that we have named Rem (regulator of export of MMTVmRNA). Rem is translated from a doubly spliced mRNA into a ca. 33 kDa protein, which is approximately two to three times larger than other retroviral export proteins. Mutations in therem open reading frame within the 3' end of the MMTV genome inhibitgag-po/ (unspliced) mRNA export from the nucleus and can be complemented by co-transfection of permissive cells with an infectious MMTV provirus or byrem complementary DMA.Moreover, the Rem C-terminus is not required for RNA export, but deletion of this domain increases export in transfection assays using an MMTV-based reporter vector. These data suggest that the C-terminus negatively regulates Rem-mediated RNA export to control MMTV structural protein production. Identification of therem gene establishes MMTV as the only murine retrovirus that encodes an auto-regulatory export protein and challenges the idea that MMTV is a simple retrovirus. To further characterize this exciting finding, we have proposed three specific aims. In the first specific aim, we will determine if Rem has specific post-translational modifications, e.g., sumoylation or phosphorylation, which affect its RNA export activity. The cell type or differentiation-specificity of such modifications will be explored. In the second specific aim, both biochemica and genetic approaches have been proposed to determine additional functions of the Rem C-terminal domain. Mutants lacking the C-terminus will be characterized for their ability to affect MMTV RNA stability, splicing, or Gag localization, processing and assembly. The Rem C-terminus also will be used in yeast two-hybrid assays and mammalian tandem affinity purifications to identify cellular proteins that may elucidate Rem functions. In the third specific aim, MMTV proviruses that lack the C-terminus of Rem will be characterized for their ability to replicate in several cell types in vitro and in vivo. These experiments may provide valuable information about novel cellular pathways required for retroviral replication and the development of mouse models for complex human retroviruses, such as HIV.

小鼠乳腺肿瘤病毒（MMTV）已被归类为一个简单的逆转录病毒，该逆转录病毒编码两个辅助蛋白，Dutpase（DU）和超抗原（SAG）。 DU蛋白和插科打蛋白酶（PR）和逆转录酶（RT）由未贴合的病毒RNA编码。简单和复杂的逆转录病毒需要病毒元素，以促进含有内含子的mRNA的核出口。简单的逆转录病毒具有直接募集参与核输出的细胞因子的C/S作用元件，而复合物逆转录病毒编码适配器蛋白，例如Rev. Rev与Viralc/S-Scoting序列结合以促进细胞出口因子募集。我们的实验表明MMTV编码第三个附件蛋白我们命名为REM（MMTVMRNA的导出器）。 REM从双重拼接中翻译 mRNA进入大约。 33 kDa蛋白，大约比其他逆转录病毒大约大约两到三倍蛋白质。 MMTV基因组抑制-PO/ Therem开放阅读框的突变（未填充）从细胞核中导出mRNA，可以通过允许细胞的共转染来补充具有感染性的MMTV病毒或BYREM互补的DMA。此外，REM C-terminus不是 RNA导出所需，但是使用该域的删除会增加转染测定法的出口基于MMTV的记者向量。这些数据表明，C末端对REM介导的负面调节 RNA导出以控制MMTV结构蛋白的产生。 Therem基因的识别建立MMTV 作为编码自动调节性出口蛋白的唯一鼠逆转录病毒，并挑战了以下观点。 MMTV是一个简单的逆转录病毒。为了进一步描述这一激动人心的发现，我们提出了三个特定的特定发现目标。在第一个特定目的中，我们将确定REM是否具有特定的翻译后修改，例如 sumoylation或磷酸化，影响其RNA输出活性。细胞类型或将探索这种修改的分化特异性。在第二个特定目的中，两个都有生物化学并提出了遗传方法来确定REM C末端的其他功能领域。缺乏C末端的突变体的特征是它们影响MMTV RNA稳定性的能力，拼接或插科打本定位，处理和组装。 REM C末端也将用于酵母两种杂交测定和哺乳动物串联亲和力净化，以鉴定可能阐明的细胞蛋白 REM功能。在第三个特定目的中，缺乏REM的C端的MMTV Provirus将是其特征是它们在体外和体内在几种细胞类型中复制的能力。这些实验可能提供有关逆转录病毒复制所需的新型细胞途径的宝贵信息开发复杂人逆转录病毒的小鼠模型，例如HIV。