Intracellular Localization of HIV-1 RNAs by Rev&Matrix

描述（由申请人提供）：人类免疫缺陷复制 1型病毒（HIV-1）涉及调节细胞内定位， 编码结构蛋白的不完全剪接的病毒mRNA。这项建议 试图更详细地了解这一规定，重点是两个病毒 蛋白质：Rev和Matrix（MA）。Rev是一种序列特异性RNA结合蛋白 促进不完全剪接的病毒在细胞质中积累， 早期的mRNAs这些HIV-1 RNA在细胞核中包埋的基础是： 人们对Rev的缺乏了解甚少，因此提出了实验来研究 这种现象。在上一个资助期间，我们发现HIV-1 Pr 55 Gag多聚蛋白的MA部分具有先前未描述的核 出口活动。虽然MA缺乏典型的富含亮氨酸的核输出 核输出信号（内斯）是通过保守的Crm 1 p途径介导的 并且在哺乳动物细胞和酵母中发挥作用。我们会继续研究 MA核输出的机制和调控及其在HIV-1中的作用 复制的我们将分析Rev和Pr 55之间的功能关系 在调节HIV-1 RNA的细胞内定位， 基因组RNA翻译或包装途径。在此前的融资 期间，我们还发现了一种破坏MA内斯（MA-M4）的突变， 将Pr 55和病毒基因组RNA错误定位到细胞核，严重损害 病毒复制Pr 55/MA-M4突变体可以以显性负性方式起作用 甚至在野生型Pr 55存在下也能错误定位基因组病毒RNA。基于 根据这些结果，我们将开发和测试一类新的显性阴性 HIV-1复制的抑制剂。最后，我们的研究结果提出了一种新的方法， 抑制任何mRNA的活性。这种“mRNA核截留” 将以HIV-1复制为模型制定和测试一项战略 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.