Post-Transcriptional Regulation of MMTV

MMTV 的转录后调控

• 批准号: 7215596
• 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/7215596
• 关键词: Address; Affect; Affinity Chromatography; Avian Leukosis; Betaretrovirus; Binding; Biochemical Genetics; Biological; Biological Assay; C-terminal; Cell Differentiation process; Cell Nucleus; Cells; Cis-Acting Sequence; Complement; Complementary DNA; Complex; Data; Development; Disease; Elements; Endopeptidases; Family member; Gagging; Genes; 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; cis acting element; dUTP pyrophosphatase; genetic regulatory protein; in vivo; insight; mRNA Export; mammary tumor virus; mouse model; mutant; novel; pol genes; research study; rev Protein; size; tissue culture; vector; viral RNA

DESCRIPTION (provided by applicant): 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 cis-acting elements that directly recruit cellular factors involved in nuclear export, whereas complex retroviruses encode adapter proteins, such as Rev. Rev binds to viral cis-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 MMTV mRNA). 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 the rem open reading frame within the 3' end of the MMTV genome inhibit gag-pol (unspliced) mRNA export from the nucleus and can be complemented by co-transfection of permissive cells with an infectious MMTV provirus or by rem complementary DNA. 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 the rem 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 biochemical 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.

描述（由申请方提供）：小鼠乳腺肿瘤病毒（MMTV）被分类为编码两种辅助蛋白（dUTR（DU）和超抗原（Sag））的简单逆转录病毒。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都需要病毒元件，以促进含内含子的mRNA的核输出。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顺式作用元件，而复杂的逆转录病毒编码衔接蛋白，例如Rev. Rev结合病毒顺式作用序列以促进细胞输出因子募集。我们的实验表明，MMTV编码的第三个辅助蛋白，我们命名为Rem（调节MMTV mRNA的出口）。Rem是从双剪接的mRNA翻译成ca。33 kDa的蛋白质，其比其他逆转录病毒输出蛋白大约2至3倍。MMTV基因组3'端内rem开放阅读框中的突变抑制gag-pol（未剪接的）mRNA从细胞核输出，并且可以通过将允许细胞与感染性MMTV前病毒共转染或通过rem互补DNA来补充。此外，Rem C-末端不是RNA输出所必需的，但是在使用基于MMT的报告载体的转染测定中，该结构域的缺失增加了输出。这些数据表明，C-末端负调控雷姆介导的RNA输出控制MMTV结构蛋白的生产。rem基因的鉴定确立了MMTV是唯一编码自动调节输出蛋白的鼠逆转录病毒，并挑战了MMTV是简单逆转录病毒的观点。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。将探索这种修饰的细胞类型或分化特异性。在第二个具体目标中，已经提出了生物化学和遗传学方法来确定Rem C-末端结构域的其他功能。缺乏C-末端的突变体将表征其影响MMTV RNA稳定性、剪接或Gag定位、加工和组装的能力。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，以鉴定可能阐明Rem功能的细胞蛋白。在第三个具体目标中，将表征缺少Rem的C-末端的MMTV前病毒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细胞途径和复杂人类逆转录病毒（如HIV）小鼠模型开发的有价值的信息。