Post-Transcriptional Regulation of MMTV

MMTV 的转录后调控

• 批准号: 7568745
• 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
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7568745
• 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

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）是一种简单的逆转录病毒，编码两个 辅助蛋白、脱氧尿苷酶（DU）和超抗原（Sag）。DU蛋白以及Gag蛋白酶（PR） 和逆转录酶（RT）由未拼接的病毒RNA编码。简单和复杂的逆转录病毒 需要促进含内含子的mRNA的核输出的病毒元件。简单逆转录病毒 具有直接募集参与核输出的细胞因子的c/s作用元件，而复杂的 逆转录病毒编码衔接蛋白，例如Rev. Rev与病毒作用序列结合，以促进 细胞输出因子的募集我们的实验表明MMTV编码第三个辅助蛋白 我们将其命名为Rem（MMTVmRNA输出调节因子）。Rem是从一个双重剪接的 mRNA进入CA。33 kDa的蛋白质，其比其他逆转录病毒输出的蛋白质大大约两到三倍。 proteins. MMTV基因组3'端开放阅读框中的突变为p53 gag-po/ （未剪接的）mRNA从细胞核输出，并且可以通过共转染允许的细胞来补充 具有感染性MMTV前病毒或byrem互补DNA。此外，Rem C-末端不是 这是RNA输出所必需的，但在使用RNA转染的转染测定中，该结构域的缺失增加了输出。 基于MMTV的报告载体。这些数据表明，C-末端负调控雷姆介导的 RNA输出以控制MMTV结构蛋白的产生。MMTV的therm基因的鉴定 作为唯一编码自动调节输出蛋白的鼠逆转录病毒， 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， 类小泛素化或磷酸化，这影响其RNA输出活性。所述细胞类型或 将探索这种修饰的分化特异性。在第二个具体目标中，生物化学 和遗传学方法已被提出来确定其他功能的雷姆C-末端 域缺乏C-末端的突变体将表征其影响MMTV RNA稳定性的能力， 拼接或Gag定位、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以鉴定可阐明 REM功能。在第三个具体目标中，将使用缺少Rem的C末端的MMTV前病毒。 其特征在于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 提供了关于逆转录病毒复制所需的新细胞途径的有价值的信息， 开发复杂的人类逆转录病毒（如HIV）的小鼠模型。