Crystallographic Studies of Retroviral Integrases

逆转录病毒，如引起AIDS的HIV-1，具有在进入宿主细胞后逆转录成线性病毒DNA的RNA基因组。将病毒DNA整合到宿主染色体中是逆转录病毒生命周期中的重要步骤，并由病毒编码的整合酶（IN）蛋白进行。逆转录病毒IN作为四聚体起作用，并催化平端病毒DNA末端的加工以及随后将这些加工的末端协同插入靶DNA的主链中。虽然IN催化反应的化学性质已经很好地理解，但对IN如何进行这些反应知之甚少。关键的未回答的问题包括：为什么病毒DNA的3 '端需要在病毒和靶DNA链连接之前被切除？四聚体IN复合物中的非催化亚基的作用是什么？两个病毒DNA末端的反应是如何协调的？逆转录病毒IN与既能整合又能切除的DNA转座酶有何不同？尽管逆转录病毒IN具有高度的医学相关性，但没有晶体或NMR结构可用于任何IN-DNA复合物或负责协调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对IN催化反应机理理解的一个重要限制。这项研究的目的是获得急需的功能性多聚体IN组装和IN-DNA复合物的三维结构信息。使用X射线晶体学，我们将确定我们已经结晶的工程化三结构域IN蛋白的结构。我们还将研究人类病原体HIV-1及其模型系统劳斯肉瘤病毒（RSV）与DNA复合的IN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抑制剂雷特格韦（raltegravir）已进入临床使用。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和设计新的抑制剂。此外，我们的研究可能有助于开发一种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，我们将专注于一个完整的3-域IN的结构测定，如我们的aim 1所提出的。

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.