Regulation of HIV-1 RNA Splicing

描述（由申请人提供）：我们的长期目标是了解HIV-1如何调节其基因产物的水平以优化病毒复制。优化HIV-1基因表达的方法之一是通过选择性RNA剪接，这是产生适当水平的不同HIV-1 mrna所必需的过程。干扰HIV-1的选择性剪接可能是一种新的抗病毒治疗方法。基本的HIV-1剪接模式在所有HIV-1毒株中都是保守的，这表明它对病毒复制的重要性。不同剪接位点的使用效率是由剪接位点本身和基因组内称为外显子剪接沉默子（ESS）和外显子剪接增强子（ESE）的顺式元件决定的。我们最近的研究结果表明，一些元素对病毒复制至关重要，因此提出了这项研究。首先，我们将在剪接位点和剪接元件中创建额外的突变，并研究它们在决定病毒Vif蛋白表达水平和病毒复制中的作用。Vif蛋白是一种使抗病毒细胞蛋白APOBEC3G失活的蛋白质。其次，我们将分离新生的HIV-1 mRNA，以确定在病毒mRNA的生命周期中剪接发生的时间点——是在RNA转录完成期间还是之后。最后，我们将研究异常组O病毒与主要组M毒株的剪接调控。与M组HIV-1株相比，O组病毒似乎使用不同的顺式元件来调节剪接。这些元素的识别可能会给我们提供关于HIV-1进化的新见解。

DESCRIPTION (provided by applicant): Our long range objective is to understand how HIV-1 regulates the levels of its gene products to optimize virus replication. One of the ways gene expression of HIV-1 is optimized is through alternative RNA splicing, a process which is necessary to produce the appropriate levels of the different HIV-1 mRNAs . Interference with HIV-1 alternative splicing may be a novel approach for antiviral therapy. The basic HIV-1 splicing pattern is conserved in all strains of HIV-1 suggesting its importance for viral replication. The efficiencies with which different splice sites are used are determined by the splice sites themselves and cis elements within the genome called exonic splicing silencers (ESS) and exonic splicing enhancers (ESE). The proposed studies are driven by our recent results indicating the crucial importance of some of the elements for virus replication. First, we will create additional mutations within splice sites and splicing elements and study their role in determining the level of expression of the viral Vif protein, a protein which acts to inactivate the antiviral cellular protein APOBEC3G, and in virus replication. Second, we will isolate nascent HIV-1 mRNA to determine at what point in the lifetime of the viral mRNA that splicing takes place-whether it is during or after RNA transcription is completed. Finally, we will study regulation of splicing of the outlier Group O viruses compared to the major Group M strains. Group O viruses appear to use different cis elements than Group M HIV-1 strains to regulate splicing. Identification of these elements may give us new insights about the evolution of HIV-1.