Crystallographic Studies of Retroviral Integrases

逆转录病毒，如导致艾滋病的HIV-1，有一个RNA基因组，当进入宿主细胞时，RNA基因组被反转录成线性病毒DNA。这种病毒DNA整合到宿主的染色体中是逆转录病毒生命周期中的一个重要步骤，由病毒编码的整合酶(IN)蛋白执行。逆转录病毒INS起四聚体的作用，催化钝端病毒DNA末端的加工，以及随后这些加工后的末端协同插入到靶DNA的骨架中。虽然人们对IN催化反应的化学性质已有很好的了解，但对IN如何进行这些反应却知之甚少。尚未回答的关键问题包括：为什么在病毒和目标DNA链连接之前需要切除病毒DNA的3‘端？四聚体IN复合体中非催化亚基的作用是什么？病毒DNA两端的反应是如何协调的？逆转录病毒INS与既能整合又能切除的DNA转座酶有什么不同？尽管逆转录病毒IN具有很高的医学相关性，但没有晶体或核磁共振结构可用于任何IN-DNA复合体或负责协同整合反应的全长三结构域IN蛋白。结构信息的缺乏一直是我们从机理上理解IN催化的反应的一个重要限制。这项拟议的研究的目标是获得急需的关于功能多聚体IN组装和IN-DNA复合体的三维结构信息。利用X射线结晶学，我们将确定我们已经结晶的工程三结构域IN蛋白质的结构。我们还将探索人类病原体HIV-1及其模型系统Rous肉瘤病毒(RSV)与DNA的复合体中INS的晶体结构。IN是一种新兴的抗HIV药物靶点，FDA批准的第一种HIV-IN抑制剂Raltegravir正在临床使用。通过这项研究获得的结构信息将有助于了解耐药突变的基础，并设计新的抑制剂。此外，我们的研究可能有助于开发具有序列特异性整合特性的工程化IN系统，以实现更安全的基因治疗。在最初的2年R21(ARRA)阶段，我们将专注于我们AIM 1中建议的完整3-结构域IN的结构确定。

Retroviruses, such as HIV-1 that causes AIDS, have an RNA genome that is reverse-transcribed into a linear viral DNA upon entering the host cell. Integration of this viral DNA into host's chromosome is an essential step in the lifecycle of retroviruses, and is carried out by the virally encoded integrase (IN) protein. Retroviral INs function as a tetramer and catalyze processing of the blunt-ended viral DNA ends as well as subsequent concerted insertions of these processed ends into the backbones of a target DNA. While the chemistry of the IN-catalyzed reactions is well understood, much less is known about how IN carries out these reactions. Key unanswered questions include; Why do the viral DNA 3'-ends need to be resected prior to the joining of viral and target DNA strands? What are the roles of non-catalytic subunits within the tetrameric IN complex? How are the reactions at two viral DNA ends coordinated? How are retroviral INs different from DNA transposases that can perform both integration and excision? Despite the high medical relevance of retroviral IN, no crystal or NMR structure is available for any IN-DNA complex or a full-length three-domain IN protein responsible for the concerted integration reaction. The lack of structural information has been a significant limitation in our mechanistic understanding of the IN-catalyzed reactions. The goal of this proposed research is to obtain the critically needed three-dimensional structural information on the functional multimeric IN assembly and IN-DNA complexes. Using x-ray crystallography we will determine the structure of an engineered three-domain IN protein that we have crystallized. We will also pursue crystal structures of INs from the human pathogen HIV-1 and its model system Rous Sarcoma Virus (RSV) in complex with DNA. IN is an emerging anti-HIV drug target, with the first FDA-approved HIV-IN inhibitor raltegravir in clinical use. The structural information obtained through this research will help understand the bases for drug-resistance mutations and design new inhibitors. In addition, our research may contribute to development of an engineered IN system with sequencing-specific integration properties for safer gene-therapy. During the initial 2-year R21 (ARRA) phase, we will focus on the structure determination of an intact 3-domain IN as proposed in our aim1.