Crystallographic studies of retroviral intasome complexes

描述(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，用于协调病毒DNA末端的插入。由HIV-1 IN形成的IN-DNA复合体是一类名为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的相互作用对于改进临床相关的抗HIV药物以及设计下一代IN抑制剂具有重要意义。最近在原型泡沫病毒(PFV)IN-DNA复合体的结构研究方面的成功使人们对逆转录病毒整合反应有了大量的见解。然而，通过X射线结晶学对来自任何其他逆转录病毒系统的IN-DNA复合体的结构表征仍然难以捉摸，这留下了一个悬而未决的问题，即在远缘逆转录病毒系统之间IN-DNA复合体的结构被保存得多好。我们假设，包括HIV-1 IN在内的规范的3-结构域IN和更大的4-结构域PFV IN中的结构域排列和IN-DNA相互作用不同，前者具有额外的DNA结合结构域和更长的结构域间连接子。为了解决这一假设，我们将确定由典型的3-结构域逆转录病毒INS形成的套体复合体的晶体结构。利用我们开发的一种新方法，我们获得了适合于结构测定的劳斯肉瘤病毒套体的晶体。我们将进一步使用我们的技术来追踪与宿主辅助因子LEDGF/p75结合的慢病毒肠套体的晶体结构。我们的X射线晶体分析将提供急需的结构信息，以更好地了解艾滋病毒-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.