HIV integrase/DNA complexes and concerted integration

描述(申请人提供)：在HIV-1感染细胞中，病毒RNA被反向转录成10kbp线性钝端DNA，随后形成细胞质前整合复合体(PIC)。PIC内的病毒整合酶(IN)在核运输之前从病毒DNA的3‘OH端切割二核苷酸。In以协调一致的方式将两个凹陷的末端插入宿主染色体，产生前病毒。细胞辅因子LEDGF/p75影响PIC在宿主染色体上选择整合位点的能力。PIC内对协调整合至关重要的IN-IN和IN-DNA相互作用以及细胞辅助因子对这一过程的影响在生化和生物物理水平上还没有很好的定义。我们最近在天然琼脂糖凝胶上发现了一种核蛋白复合体，其中IN非共价并列在病毒DNA的两端，产生与PIC相关的突触复合体(SC)。我们将研究SC的组装性质，研究IN保护末端U5和U3 DNA序列免受DNaseI消化的能力，并确定位于SC中的化学交联的二聚体、四聚体和较大尺寸的IN多聚体的摩尔比。在SC中，IN独特的~32BP DNaseI保护足迹表明与更大尺寸的多聚体存在结构关系。我们将研究这些交联物种之间的关系，以确定负责形成四聚体的独特的亚基间残基，这是通过对交联的胰蛋白酶二肽的质谱学研究确定的。凝胶内荧光、共振能量转移衍生距离测量和原子力显微镜将确定SC内病毒DNA末端的拓扑。我们期望生成一个描述SC及其与PIC关系的结构范式。我们将定义细胞辅助因子LEDGF/p75与SC的物理相互作用，并通过测量SC中荧光标记的DNA底物之间的距离来确定它是否影响SC。我们将开发一个模型系统来确定LEDGF/p75是否影响显色超螺旋靶DNA上的宿主位点选择以进行协同整合。我们将在体外研究已知的II类IN突变体，这些突变体具有催化活性并具有核后进入缺陷。我们还将确定在SC中观察到的交联型IN四聚体中识别的唯一残基的功能性。这些IN残基的定点突变将用于在体内探测PIC和在体外探测SC。从R21研究中获得的知识将被用来进一步研究PIC和细胞辅助因子在整合中的参与。R21/R33研究的完成将促进对体外和体内协同整合的必要和可实现的理解。与公共卫生相关：我们正在建立一个结构性信息平台，以了解能够协调整合的HIV-1突触复合体(SC)。我们将确定SC中IN亚基的相互作用，以确定哪些残基负责IN四聚体的形成。这些残基的功能将通过定点突变来研究，以探索体内的PIC和体外的SC。我们将确定LEDGF/p75是否与SC物理相互作用，并测试它是否影响含有HIV-1宿主位点共识序列的显色超螺旋靶DNA上的宿主位点选择。

DESCRIPTION (provided by applicant): In HIV-1 infected cells, the viral RNA is reverse transcribed into a 10 kbp linear blunt-ended DNA followed by the formation of the cytoplasmic preintegration complex (PIC). The viral integrase (IN) within the PIC cleaves a dinucleotide from the 3' OH ends of the viral DNA prior to nuclear transport. IN inserts the two recessed ends in a concerted fashion into the host chromosome, producing the provirus. The cellular co- factor, LEDGF/p75, influences the ability of the PIC to select sites on the host chromosomes for integration. The IN-IN and IN-DNA interactions within the PIC critical for concerted integration and the effect of cellular co-factors on this process are not well defined at the biochemical and biophysical levels. We recently identified a nucleoprotein complex on native agarose gels where IN non-covalently juxtaposes two viral DNA ends that produces the synaptic complex (SC) that possesses properties associated with the PIC. We will study the assembly properties of the SC, investigate the ability of IN to protect the terminal U5 and U3 DNA sequences from DNaseI digestion and determine the molar ratios of chemically cross-linked dimers, tetramers and a larger-size multimer of IN located in the SC. A unique ~32 bp DNaseI protective footprint by IN in the SC suggests a structural relationship to the larger-size multimer. We will investigate the relationships between these cross-linked species to identify the unique inter-subunit residues responsible for formation of the tetramer, as determined by mass spectrometry studies of cross-linked tryptic dipeptides. In- gel fluorescence resonance energy transfer-derived distance measurements and atomic force microscopy will determine the topology of the two viral DNA ends within the SC. We expect to generate a structural paradigm describing the SC and its relationship to the PIC. We will define the cellular co-factor LEDGF/p75 physical interactions with the SC and determine if it affects the SC by measuring the distance between fluorophore- labeled DNA substrates in the SC. We will develop a model system to determine if LEDGF/p75 influences host-site selection on chromatinized supercoiled target DNA for concerted integration. We will study known Class II IN mutants in vitro that are catalytically active and possess post-nuclear entry defects. We will also determine the functionality of the unique residues identified in the cross-linked IN tetramer observed in the SC. Site-directed mutagenesis of these IN residues will be performed for probing of the PIC in vivo and the SC in vitro. The knowledge gain from the R21 studies will be used to further investigate the PIC and the involvement of cellular co-factors in integration. Completion of the R21/R33 studies will foster a necessary and achievable understanding of concerted integration both in vitro and in vivo. PUBLIC HEALTH RELEVANCE: We are building a platform of structural information to understand the HIV-1 synaptic complex (SC) capable of concerted integration. We will determine the IN subunit interactions within SC to define what residues are responsible for formation of the IN tetramer. The functionality of these residues will be investigated by site-directed mutagenesis to probe the PIC in vivo and the SC in vitro. We will determine if LEDGF/p75 physically interacts with SC and test whether it influences host-site selection on chromatinized supercoiled target DNA that contains HIV-1 host-site consensus sequences.