Post-Transcriptional Regulation of MMTV

描述(申请人提供)：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)都是由病毒非剪接RNA编码的。无论是简单的还是复杂的逆转录病毒，都需要病毒成分来促进含有内含子的mRNAs的核输出。简单的逆转录病毒具有直接招募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适配蛋白，如REV结合到病毒的顺式作用序列，以促进细胞输出因子的招募。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(MMTV mRNA输出调节因子)。REM从一个双剪接的mRNA翻译成一个约33 kDa的蛋白，大约是其他逆转录病毒输出蛋白的两到三倍。MMTV基因组3‘端rem开放阅读框的突变抑制了Gag-Poll(未剪接)mRNA从细胞核输出，并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，RemC末端不是RNA输出所必需的，但该结构域的缺失会增加使用基于MMTV的报告载体的转染检测中的输出。这些数据表明，C末端负调控Rem介导的RNA输出，以控制MMTV结构蛋白的生产。对rem基因的鉴定使MMTV成为唯一一种编码自我调节输出蛋白的小鼠逆转录病毒，并挑战了MMTV是一种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。在第一个特定目标中，我们将确定Rem是否具有影响其RNA输出活性的特定翻译后修饰，例如SUMO化或磷酸化。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，已经提出了生化和遗传学方法来确定Rem C-末端结构域的额外功能。缺乏C末端的突变体将因其影响MMTV RNA稳定性、剪接或Gag定位、加工和组装的能力而被表征。Rem C末端还将用于酵母双杂交分析和哺乳动物的串联亲和纯化，以鉴定可能阐明Rem功能的细胞蛋白。在第三个特定目标中，缺乏Rem C末端的MMTV前病毒将因其在体外和体内几种细胞类型中复制的能力而被表征。这些实验可能提供有关逆转录病毒复制和开发复杂人类逆转录病毒(如HIV)的小鼠模型所需的新细胞途径的有价值的信息。

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.