Intracellular Localization of HIV-1 RNAs by Rev&Matrix

描述(申请人提供)：复制人类免疫缺陷 1型病毒(HIV-1)涉及对细胞内定位的调节 编码结构蛋白的未完全剪接的病毒mRNAs。这项建议 寻求更详细地了解这一规定，重点是两个病毒 蛋白质：REV和基质(MA)。REV是一种序列特异性的RNA结合蛋白 这有助于未完全剪接的病毒在细胞质内积累 早期的mRNAs。这些HIV-1 RNA被核包裹的基础是 人们对REV的缺失知之甚少，并提议用实验进行研究 这种现象。在之前的资助期间，我们发现HIV-1 Pr55Gag多聚蛋白的mA部分具有一个以前未描述的核 出口活动。尽管MA缺乏典型的富含亮氨酸的核出口 信号(NES)，核输出是通过保守的CRM1P途径介导的 在哺乳动物细胞和酵母中都有功能。我们会继续研究 MA核输出的机制、调控及其在HIV-1中的作用 复制。我们将分析Rev和Pr55之间的函数关系 在调节HIV-1RNA在细胞内的定位和在引导病毒 基因组RNA到翻译或包装的途径。比之前的资金 期间我们还发现了一种破坏MA NES的突变(MA-M4) 将Pr55和病毒基因组RNA错误定位到细胞核，严重危害 病毒复制。Pr55/MA-M4突变体可以以显性-负性方式起作用 使基因组病毒RNA错位，即使在野生型Pr55存在的情况下也是如此。基座 在这些结果的基础上，我们将开发和测试一类新的显性否定 艾滋病毒-L复制的抑制剂。最后，我们的结果提出了一种新的方法。 用于抑制任何信使核糖核酸的活性。这种“信使核糖核酸的核捕获” 将以艾滋病毒-1复制为模型制定和测试战略 系统。

DESCRIPTION (provided by applicant): Replication of human immunodeficiency virus type 1 (HIV-1) involves regulation of the intracellular localization of incompletely spliced viral mRNAs that encode structural proteins. This proposal seeks to understand this regulation in greater detail, focusing on two viral proteins: Rev and Matrix (MA). Rev is a sequence-specific RNA binding protein that facilitates the cytoplasmic accumulation of incompletely spliced viral mRNAs at early times. The basis for nuclear entrapment of these HIV-1 RNAs in the absence of Rev is poorly understood and experiments are proposed to study this phenomenon. Over the previous funding period we discovered that the HIV-1 MA moiety of the Pr55 Gag polyprotein has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal (NES), nuclear export is mediated through the conserved Crm 1p pathway and functions in both mammalian cells and yeast. We will continue to study the mechanism and regulation of MA nuclear export, and its role in HIV-1 replication. We will analyze the functional relationship between Rev and Pr55 in regulating HIV-1 RNA intracellular localization, and in directing viral genomic RNA to translation or packaging pathways. Over the previous funding period we also found that a mutation that disrupts the MA NES (MA-M4) mislocalizes Pr55 and viral genomic RNA to the nucleus, severely compromising viral replication. The Pr55/MA-M4 mutant can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild type Pr55. Based upon these results, we will develop and test a new class of dominant-negative inhibitors of HIV-l replication. Finally, our results suggest a novel method for inhibiting the activity of any mRNA. This "mRNA nuclear entrapment" strategy will be developed and tested using HIV-1 replication as a model system.