Post-Transcriptional Regulation of MMTV

小鼠乳腺肿瘤病毒(MMTV)是一种简单的逆转录病毒，编码两种 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)是由非剪接病毒RNA编码的。简单逆转录病毒和复杂逆转录病毒 要求病毒成分促进含有内含子的mRNAs的核出口。简单逆转录病毒 有c/S作用元件直接招募参与核出口的细胞因子，而复杂 逆转录病毒编码适配蛋白，如Rev.REV结合到病毒C/S作用序列，以便于 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 我们已经命名为Rem(MMTVmRNA出口监管机构)。REM从双剪接体翻译而来 MRNA转化为约33 kDa的蛋白质，大约是其他逆转录病毒出口的两到三倍 蛋白质。MMTV基因组抑制物3‘端开放阅读框的突变 (未剪接的)mRNA从细胞核输出，并可通过共转染允许细胞来补充 具有感染性的MMTV前病毒或BREM互补DMA。此外，REM C末端不是 RNA输出所必需的，但删除该结构域会增加使用 基于MMTV的记者载体。这些数据表明，C-末端负向调节Rem介导的 RNA出口以控制MMTV结构蛋白的生产。对该基因的鉴定建立了MMTV 作为唯一一种编码自动调节出口蛋白的小鼠逆转录病毒，并挑战了 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是否有特定的翻译后修饰，例如， 苏莫化或磷酸化，影响其RNA输出活性。单元格类型或 将探索这种修饰的差异性-特异性。在第二个具体目标中，生物化学和 并且已经提出了遗传方法来确定Rem C-末端的附加功能 域。缺乏C末端的突变体将因其影响MMTV RNA稳定性的能力而被表征， 拼接，或插口定位，加工和组装。Rem C-末端也将用于酵母 双杂交分析和哺乳动物串联亲和纯化鉴定可能阐明 REM功能。在第三个具体目标中，缺少Rem的C末端的MMTV提供将是 其特点是能够在体外和体内的几种细胞类型中复制。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和 开发复杂的人类逆转录病毒的小鼠模型，如艾滋病毒。

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.