Crystallographic studies of retroviral intasome complexes

描述（由申请人提供）：逆转录病毒整合是由病毒整合酶（IN）催化的，它与线性病毒DNA基因组的两端形成四聚体复合体，并捕获细胞靶DNA以协调插入病毒DNA末端。由HIV-1 IN形成的IN- dna复合体是一类称为IN链转移抑制剂的抗病毒药物的靶标。因此，更好地了解IN- dna相互作用对于改善临床相关的抗hiv药物以及设计下一代IN抑制剂非常重要。最近在原型泡沫病毒（PFV） in - dna复合体的结构研究中取得的成功导致了对逆转录病毒整合反应的大量见解。然而，通过x射线晶体学对来自任何其他逆转录病毒系统的IN-DNA复合物的结构表征仍然难以捉摸，这留下了一个问题，即IN-DNA复合物的结构在远亲逆转录病毒系统之间的保守程度如何。我们假设，在包括HIV-1在内的典型3结构域内，包括HIV-1在内的典型3结构域内的结构域排列和IN-DNA与病毒和靶DNA分子的相互作用与具有额外DNA结合结构域和更长的结构域间连接体的更大的4结构域PFV内的结构域不同。为了解决这一假设，我们将确定由典型的3域逆转录病毒蛋白形成的侵入体复合物的晶体结构。利用我们开发的一种新方法，获得了适合于结构测定的劳斯肉瘤病毒侵入体晶体。我们将进一步利用我们的技术来研究结合宿主辅助因子LEDGF/p75的慢病毒侵入体的晶体结构。我们的x射线晶体学分析将提供急需的结构信息，以更好地了解HIV-1和密切相关的逆转录病毒的整合。

DESCRIPTION (provided by applicant): Retrovirus integration is catalyzed by the viral integrase (IN), which forms a tetrameric complex with both ends of the linear viral DNA genome and captures a cellular target DNA for concerted insertions of the viral DNA termini. The IN-DNA complex formed by HIV-1 IN is the target of a class of antiviral drugs called IN strand- transfer inhibitors. A better understanding of the IN-DNA interactions is therefore important for improving the clinically relevant anti-HIV drugs as well as designing next-generation IN inhibitors. The recent success in the structural studies of prototype foamy virus (PFV) IN-DNA complexes led to tremendous amount of insights into the retrovirus integration reaction. However, structural characterization of IN-DNA complexes from any other retrovirus systems by X-ray crystallography has remained elusive, leaving open the question as to how well the architecture of the IN-DNA complexes is conserved between distantly related retrovirus systems. We hypothesize that domain arrangement and IN-DNA interactions within the intasome, a nucleoprotein complex containing the tetramer of IN with the viral and target DNA molecules, are different between the canonical 3- domain IN including HIV-1 IN and the larger 4-domain PFV IN featuring an additional DNA-binding domain and longer inter-domain linkers. To address this hypothesis, we will determine crystal structures of the intasome complexes formed by the canonical 3-domain retroviral INs. Using a novel approach we have developed, crystals of the Rous sarcoma virus intasome suitable for structure determination have been obtained. We will further use our technique to pursue the crystal structure of a lentiviral intasome bound to the host co-factor LEDGF/p75. Our X-ray crystallographic analyses will provide the critically needed structural information to better understand the integration of HIV-1 and closely related retroviruses.