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）被归类为一种简单的逆转录病毒，编码两个辅助蛋白、dUTPase (DU) 和超抗原 (Sag)。 DU 蛋白以及 Gag、蛋白酶 (PR) 和逆转录酶（RT）由未剪接的病毒RNA编码。简单和复杂的逆转录病毒需要促进含内含子 mRNA 核输出的病毒元件。简单逆转录病毒具有顺式/顺式作用元件，直接招募参与核输出的细胞因子，而复杂逆转录病毒编码接头蛋白，例如Rev。Rev结合到病毒c/s作用序列以促进细胞输出因子招募。我们的实验表明 MMTV 编码第三种辅助蛋白我们将其命名为 Rem（MMTVmRNA 输出调节器）。 Rem 翻译自双拼接 mRNA 转化为 ca。 33 kDa 蛋白质，大约是其他逆转录病毒输出的两到三倍蛋白质。 MMTV 基因组抑制gag-po/ 3'端内的rem开放阅读框突变（未剪接的）mRNA 从细胞核输出，可以通过允许细胞的共转染来补充具有传染性 MMTV 原病毒或 byrem 互补 DMA。此外，Rem C 末端不是 RNA 输出所需，但删除该结构域会增加使用转染测定中的输出基于 MMTV 的记者载体。这些数据表明 C 末端对 Rem 介导的负调控 RNA 输出控制 MMTV 结构蛋白的产生。鉴定rem基因建立MMTV 作为唯一编码自动调节输出蛋白的鼠逆转录病毒，并挑战了以下观点： MMTV 是一种简单的逆转录病毒。为了进一步描述这一令人兴奋的发现，我们提出了三个具体的目标。在第一个具体目标中，我们将确定 Rem 是否具有特定的翻译后修饰，例如，苏酰化或磷酸化，影响其 RNA 输出活性。细胞类型或将探讨此类修饰的分化特异性。在第二个具体目标中，生物化学已经提出了遗传方法来确定 Rem C 末端的其他功能领域。缺乏 C 末端的突变体将以其影响 MMTV RNA 稳定性的能力为特征，拼接，或 Gag 本地化、加工和组装。 Rem C 末端也将用于酵母双杂交测定和哺乳动物串联亲和纯化来鉴定可阐明的细胞蛋白雷姆函数。在第三个具体目标中，缺少 Rem C 末端的 MMTV 原病毒将被其特征在于它们能够在体外和体内在多种细胞类型中复制。这些实验可能提供有关逆转录病毒复制所需的新细胞途径的有价值的信息开发复杂人类逆转录病毒（例如艾滋病毒）的小鼠模型。