Structural Control of Human Co-factors for Retroviral Gene Expression

 描述(申请人提供)：在这个项目中，我们将剖析关键宿主辅因子TAT-SF1在人类与HIV-1RNA剪接之间的活性。复杂的逆转录病毒，如HIV-1，在病毒生命周期的早期和后期，利用人类剪接体机制紧密协调其剪接的mRNA和基因组RNA的生产。目前，HIV-1通过宿主剪接循环主导进展的分子机制仍然是该领域的突出问题。TAT-SF1是一种宿主蛋白，在逆转录病毒生命周期的早期阶段，对产生完全剪接的HIV-1mRNAs至关重要。塔特 已知SF1与剪接体的U2小核核糖核蛋白亚基(SnRNP)相关，并调节特定人类转录本的剪接。因此，对TAT-SF1的研究将对了解宿主与病毒的相互作用非常重要，从而为开发新的生化工具或剪接水平的治疗干预方法奠定基础。目前，TAT-SF1在HIV-1和人类剪接中的潜在结构和功能作用尚不清楚。我们将通过涉及多学科方法的强有力的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。具体地说，我们将确定人类U2 SnRNA和U2 SnRNP亚基之间的TAT-SF1相互作用，并测试这些接口在人类剪接与HIV-1剪接和传染性方面的功能。我们还将研究HIV-1预防人类剪接机制的一种新的作用机制，在该机制中，早期HIV-1Rev蛋白通过破坏宿主TAT-SF1-U2 SnRNP复合体促进晚期未剪接的HIV-1 RNA。我们已经：(I)制备了TAT-SF1 RNA识别基序的衍射晶体，(Ii)获得了TAT-SF1与U2 SnRNP的SF3b155亚基之间的复合物的初步结构，以及(Iii)在人类细胞系中产生了稳定的TAT-SF1的击倒。此外，我们的初步结果表明，HIV-1Rev蛋白与人TAT-SF1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，拟议中的实验的成功完成将为针对宿主辅因子的新治疗策略奠定基础，从而绕过困扰现有抗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.