Structural Control of Human Co-factors for Retroviral Gene Expression

 描述（由申请人提供）：在本项目中，我们将分析关键宿主辅因子Tat-SF 1在人类与HIV-1 RNA剪接中的活性。复杂的逆转录病毒，如HIV-1，在病毒生命周期的早期和晚期，利用人类剪接体机制紧密协调产生其剪接的mRNA和基因组RNA。目前，负责HIV-1通过宿主剪接循环的主要进展的分子机制仍然是该领域的突出问题。Tat-SF 1是一种宿主蛋白，在逆转录病毒生命周期的早期阶段，它在完全剪接的HIV-1 mRNA的产生中起关键作用。达特 已知SF 1与剪接体的U2小核核糖核蛋白亚基（snRNP）结合并调节特定人类转录物的剪接。因此，Tat-SF 1的研究对于理解宿主-病毒相互作用将是重要的，从而为在剪接水平上开发新的生化工具或治疗干预方法奠定基础。目前，Tat-SF 1在HIV-1和人类剪接中的潜在结构和功能作用仍然未知。我们将通过涉及多学科方法的强有力合作来解决这一知识差距，这些方法包括前mRNA剪接因子的结构生物化学，分子生物学和病毒学。具体来说，我们将确定达特-SF 1之间的相互作用的人U2 snRNA和U2 snRNP亚基，并测试这些接口的功能，在人类剪接与HIV-1剪接和感染性。我们还将研究HIV-1抢先人类剪接机制的新作用机制，其中早期HIV-1 Rev蛋白通过破坏宿主Tat-SF 1-U2 snRNP复合物促进晚期未剪接的HIV-1 RNA。我们已经有：（i）产生Tat-SF 1 RNA识别基序的衍射晶体，（ii）获得Tat-SF 1和U2 snRNP的SF 3b 155亚基之间的复合物的初始结构，和（iii）在人细胞系中产生Tat-SF 1的稳定敲低。此外，我们的初步结果表明，HIV- 1 Rev蛋白与人Tat-SF 1相关。除了阐明HIV-1操纵宿主基因表达途径的新作用机制外，拟议实验的成功完成将为靶向宿主辅因子的新治疗策略奠定基础，从而规避困扰现有抗HIV-1治疗的耐药性挑战。

 DESCRIPTION (provided by applicant): In this project, we will dissect the activity of key host cofactor Tat-SF1 in human versus HIV-1 RNA splicing. Complex retroviruses such as HIV-1 co-opt the human spliceosome machinery for tightly coordinated production of their spliced mRNAs and genomic RNAs during the early and late stages of the viral life cycle. At present, the molecular mechanisms responsible for the dominant progression of HIV-1 through the host splicing cycle remain outstanding questions in the field. Tat-SF1 is a host protein that is critica in the production of fully spliced HIV-1 mRNAs in the early stage of the retroviral life cycle. Tat SF1 is known to associate with the U2 small nuclear ribonucleoprotein subunit (snRNP) of the spliceosome and regulate the splicing of specific human transcripts. As such, studies of Tat-SF1 will be important to understand host-virus interactions, thereby laying the groundwork for the development of new biochemical tools or therapeutic intervention approaches at the level of splicing. At present, the underlying structural and functional roles of Tat-SF1 in HIV-1 and human splicing remain unknown. We will address this knowledge gap by a strong collaboration involving multidisciplinary approaches that include structural biochemistry of pre-mRNA splicing factors, molecular biology, and virology. Specifically, we will determine Tat- SF1 interactions among the human U2 snRNA and U2 snRNP subunits, and test these interfaces for functions in human splicing versus HIV-1 splicing and infectivity. We also will investigate a new mechanism of action for HIV-1 to pre-empt the human splicing machinery, in which the early stage HIV-1 Rev protein promotes late stage unspliced HIV-1 RNAs by disrupting the host Tat-SF1-U2 snRNP complex. Already we have: (i) produced diffracting crystals of the Tat-SF1 RNA recognition motif, (ii) obtained an initial structure of a complex between the Tat-SF1 and the SF3b155 subunit of the U2 snRNP, and (iii) generated a stable knockdown of Tat-SF1 in a human cell line. Moreover, our preliminary results demonstrate that the HIV- 1 Rev protein associates with human Tat-SF1. In addition to elucidating new mechanisms of action for HIV-1 to manipulate host pathways of gene expression, successful completion of the proposed experiments will set the stage for new therapeutic strategies that target a host cofactor, thereby circumventing drug resistance challenges that plague existing anti-HIV-1 treatments.