Crystallographic Studies of Retroviral Integrases

逆转录病毒，如导致艾滋病的HIV-1，其RNA基因组在进入宿主细胞后被逆转录成线性病毒DNA。将病毒DNA整合到宿主染色体中是逆转录病毒生命周期中必不可少的一步，由病毒编码的整合酶（in）蛋白完成。逆转录病毒蛋白作为四聚体发挥作用，催化钝端病毒DNA末端的加工，以及随后将这些加工过的末端协同插入目标DNA的主干。虽然IN催化反应的化学性质已经被很好地理解，但对于IN如何进行这些反应却知之甚少。关键的未解问题包括；为什么病毒DNA的3'端需要在病毒和目标DNA链连接之前被切除？非催化亚基在四聚体IN配合物中的作用是什么？两个病毒DNA末端的反应是如何协调的？逆转录病毒INs与DNA转座酶有何不同？尽管逆转录病毒IN具有很高的医学相关性，但没有任何IN- dna复合物或负责协同整合反应的全长三结构域IN蛋白的晶体或核磁共振结构。结构信息的缺乏是我们对in催化反应机理理解的一个重大限制。本研究的目的是获得功能性多聚体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三域IN蛋白的结构。我们还将研究来自人类病原体HIV-1及其模型系统Rous肉瘤病毒（RSV）的INs与DNA复合物的晶体结构。IN是一种新兴的抗hiv药物靶点，fda批准的第一个HIV-IN抑制剂雷替格拉韦在临床使用。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和设计新的抑制剂。此外，我们的研究可能有助于开发具有测序特异性整合特性的工程In系统，以实现更安全的基因治疗。在最初的2年R21 （ARRA）阶段，我们将专注于我们的目标中提出的完整3-domain 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.