BIOCHEMICAL MECHANISM OF HIV DNA INTEGRATION

人类免疫缺陷病毒1型(HIV-1)必须将其RNA基因组的cDNA拷贝整合到受感染的细胞染色体中，才能有效复制。在整合中的关键病毒角色是反式作用整合酶(IN)蛋白，它作为病毒的一部分进入细胞，以及顺式作用DNA附着(ATT)位点，由反转录产生的线状DNA末端组成。在细胞中，整合是通过大的亚病毒核蛋白前整合复合体(PIC)介导的，这些复合体来自感染病毒粒子的核心。从感染细胞中分离出的PICs在体外可以将其内源DNA整合到添加的靶DNA中。此前，照片因其尺寸较大而被提纯。单独施胶并不是一种非常有效的净化技术：它本身就是稀释的，而且大的细胞集合体与PIC结合在一起。将开发基于亲和层析的新技术，将PIC的纯度和产率提高约100倍于现有技术。除IN外，宿主因子在逆转录病毒PIC的结构和功能中起着重要作用。两种宿主蛋白，HMG I(Y)和屏障自整合因子(BAF)，以前被认为与HIV-1整合有关，但尚不清楚其中哪一种与生理相关。在纯化的PIC中将测定HMG I(Y)和BAF对IN和cDNA的化学计量比。其他病毒蛋白，包括基质、逆转录酶、VPR和与HIV-1 PIC协同作用的核衣壳蛋白。虽然不需要DNA重组，但基质和VPR可能会促进未分裂细胞中PIC的核输入。这些病毒蛋白的化学计量比将在纯化的样本中确定，以及IN、基质和逆转录酶的折叠拓扑结构。目前对PIC结构和功能起重要作用的蛋白质-蛋白质相互作用模型还没有很好的定义，但预测在体内IN催化的两个cDNA端是偶联的。初步数据支持这样一种模型，即在最初的催化步骤中，cdna末端是解偶联的，然后通过蛋白质-蛋白质相互作用聚集在一起进行整合。病毒和宿主因素对这些相互作用的贡献将被确定。一种新的IN突变体被鉴定出，除了正常的整合外，它还促进一次只有一个cDNA端的整合，这是一种有害于病毒生长的途径。将对这些突变PIC进行详细分析，以确定正常PIC功能所需的蛋白质-蛋白质和蛋白质-DNA相互作用。这些实验的结果将被用来制定HIV-1 PIC的结构和功能组织的详细模型，这将有助于针对HIV-1整合的抗病毒药物的设计。

Human immunodeficiency virus type 1 (HIV-1) must integrate the cDNA copy of its RNA genome into an infected cell chromosome in order to efficiently replicate. The key viral players in integration are the trans-acting integrase (IN) protein, which enters the cell as a part of the virus, and the cis-acting DNA attachment (att) site, comprised of the ends of linear cDNA made by reverse transcription. In cells, integration is mediated through large subviral nucleoprotein preintegration complexes (PICs) that are derived from the cores of infecting virions. PICs isolated from infected cells can integrate their endogenous cDNA into an added target DNA in vitro. PICs were previously purified based on their large size. Sizing on its own is not a very efficient purification technique: it is inherently diluting, and large cellular assemblies copurify with PICs. Novel techniques based on affinity chromatography will be developed to increase the purity and yield of PICs approximately 100-fold over existing techniques. In addition to IN, host factors play essential roles in the structure and function of retroviral PICs. Two host proteins, HMG I(Y) and the barrier-to-autointegration factor (BAF), were previously implicated in HIV-1 integration, but it unclear which of these is physiologically-relevant. The stoichiometries of HMG I(Y) and BAF to IN and cDNA will be determined in purified PICs. Other viral proteins, including matrix, reverse transcriptase, Vpr, and nucleocapsid cofractionate with HIV-1 PICs. Although not needed for DNA recombination, matrix and Vpr may facilitate the nuclear import of PICs in nondividing cells. The stoichiometries of these viral proteins will be determined in purified samples, along with the folding topologies of IN, matrix and reverse transcriptase. Current models of protein-protein interactions important for PIC structure and function are not well defined, but predict the two cDNA ends are coupled for IN catalysis in vivo. Preliminary data supports a model where the cDNA ends are uncoupled for an initial catalytic step, and then come together through protein-protein interactions for integration. The contribution of viral and host factors to these interactions will be determined. A novel IN mutant was identified that in addition to normal integration promotes the integration of just one cDNA end at a time, a pathway that is deleterious to virus growth. These mutant PICs will be analyzed in detail to identify protein-protein and protein-DNA interactions required for normal PIC function. The results of these experiments will be used to formulate a detailed model of the structural and functional organization of HIV-1 PICs, which will aid the design of antiviral drugs targeted against HIV-1 integration.