Retroviral Integration

逆转录病毒整合

• 批准号: 8349018
• 负责人: stephen h hughes
• 金额: $ 123.81万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349018
• 关键词: Active Sites; Affect; Binding; Cells; Chimeric Proteins; Chromatin; Chromosome inversion; Chromosomes; Collaborations; Complex; Cultured Cells; DNA; DNA Insertion Elements; DNA Integration; Development; Disease; Dose; Enzymes; Event; Generations; Genes; Genome; Goals; HIV; HIV Drug Resistance Program; HIV-1; Histones; Human; In Vitro; Insertional Activations; Integrase; Integrase Inhibitors; Knockout Mice; Laboratories; Lead; Length; Life; Ligand Binding Domain; Malignant Neoplasms; Maps; Mediating; Mus; Mutate; Mutation; N Domain; Nature; Nucleotides; Oncogenes; Patients; Pharmaceutical Preparations; Problem Solving; Process; Protein Binding; Protein Binding Domain; Proteins; Reaction; Recombinants; Reporting; Resistance; Resolution; Reverse Transcription; Sequence-Specific DNA Binding Protein; Site Visit; Specificity; Spumavirus; Structure; System; Tail; Technology; Tertiary Protein Structure; Testing; Tissues; Toxic effect; Viral; Viral Genome; Work; base; design; gene therapy; inhibitor/antagonist; lens epithelium-derived growth factor; research study; vector; viral DNA; Active Sites; Affect; Binding; Cells; Chimeric Proteins; Chromatin; Chromosome inversion; Chromosomes; Collaborations; Complex; Cultured Cells; DNA; DNA Insertion Elements; DNA Integration; Development; Disease; Dose; Enzymes; Event; Generations; Genes; Genome; Goals; HIV; HIV Drug Resistance Program; HIV-1; Histones; Human; In Vitro; Insertional Activations; Integrase; Integrase Inhibitors; Knockout Mice; Laboratories; Lead; Length; Life; Ligand Binding Domain; Malignant Neoplasms; Maps; Mediating; Mus; Mutate; Mutation; N Domain; Nature; Nucleotides; Oncogenes; Patients; Pharmaceutical Preparations; Problem Solving; Process; Protein Binding; Protein Binding Domain; Proteins; Reaction; Recombinants; Reporting; Resistance; Resolution; Reverse Transcription; Sequence-Specific DNA Binding Protein; Site Visit; Specificity; Spumavirus; Structure; System; Tail; Technology; Tertiary Protein Structure; Testing; Tissues; Toxic effect; Viral; Viral Genome; Work; base; design; gene therapy; inhibitor/antagonist; lens epithelium-derived growth factor; research study; vector; viral DNA

a. Integration of DNAs with aberrant ends. These experiments grew out of experiments designed to examine how mutations either in RT or in the viral genome altered the process of reverse transcription; this led to the generation of linear viral DNAs with a high proportion of aberrant ends. Having characterized the nature of the errors at the ends of the viral DNAs, we then examined how the aberrant ends affected viral DNA integration. Using the RCAS vector system to facilitate the rescue of aberrant-end viral DNAs, we found that (1) integration is not concerted; if a viral DNA has a good end and an aberrant end, the good end integrates using viral integrase (IN) and the aberrant end is integrated with high efficiency, apparently by host enzymes. (2) In most, but not all, cases, microhomology is involved in the host-mediated integration reaction. (3) The host integration reaction frequently causes a relatively large duplication (hundreds to thousands of nucleotides), and less frequently a deletion of host sequences. (4) The host joining reaction usually, but not always, involves the loss of viral sequences. (5) More rarely, complex events occur that involve duplications of viral sequences, inversion of host sequences, or the insertion of sequences from different chromosomes. These experiments may have implications for the treatment of patients with suboptimal doses of IN inhibitors; suboptimal therapy may lead to aberrant integrations. b. Developing IN inhibitors. Dr. Y. Pommier is testing compounds developed by Dr. T. Burke for their ability to inhibit HIV integration in vitro (using purified recombinant IN); we are testing their effect on viral replication and their toxicity in cultured cells. Until quite recently, it was not possible to use structural information to guide the development of IN inhibitors. However, Dr. P. Cherepanov and his colleagues have obtained high-resolution structures of full-length foamy virus (FV) IN in complexes with both DNA substrates and anti-IN drugs. Dr. Cherepanov has joined our collaboration and will solve the structures of FV IN in complex with some of the more promising compounds developed by Dr. Burke. The FV active site is similar, but not identical, to the HIV-1 active site, so the FV structures should be useful. However, it should be possible to mutate the region around the FV active site to make it more similar to HIV-1 IN, which should provide better guidance for design/development of more effective inhibitors. c. Redirecting the integration of HIV-1 DNA. Redirecting HIV-1 DNA integration has the potential to help make gene therapy safer because it may help solve the problems associated with the insertional activation of oncogenes; in addition, the technology can be used to determine where on the genome proteins/domains bind to chromatin. Lens epithelium derived growth factor (LEDGF) interacts with HIV-1 IN, which directs HIV-1 DNA integration to the bodies of expressed genes. The C-terminus of LEDGF contains an IN-binding domain and the N-terminus binds chromatin. We and others showed that replacing the N-terminus of LEDGF with chromatin-binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. The initial experiments were done either with single CBDs, or, in one case, two domains from a larger protein. We will do a number of additional experiments: (1) We will investigate how multiple CBDs interact to define the specificity with which proteins bind chromatin. Ultimately, we will determine how whole proteins bind chromatin. We will obtain well-characterized domains/proteins from the laboratory of our collaborator Dr. C.D. Allis. (2) All the domains we have analyzed so far bind marks on histone tails. We have started to apply the technology to proteins that bind specific DNA sequences, and will investigate domains/proteins that interact with chromatin in other ways. (3) Genes are expressed differently in different tissues and in certain disease states, like cancer. This implies a change in the chromatin. We have just obtained, from our collaborator Dr. A. Engelman, LEDGF knockout mice, and will use cells/tissues from these mice to determine how differentiation and transformation affect the distribution of some well-characterized CBD-IBD fusions. (4) We will adapt the technology to human cells. [Corresponds to Hughes Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]

一个。 DNA与异常末端的整合。这些实验源于旨在检查RT或病毒基因组中突变如何改变逆转录过程的实验。这导致了线性病毒DNA的产生，并具有很高的异常末端。在表征了病毒DNA末端的误差的性质之后，我们检查了异常末端如何影响病毒DNA的整合。使用RCAS矢量系统促进挽救异常的病毒DNA，我们发现（1）集成没有协调；如果病毒DNA具有良好的末端和异常端，则良好的末端使用病毒积分酶（IN）积分，并且异常端的效率高显着，显然是由宿主酶集成的。 （2）在大多数但不是所有情况下，微型学都参与宿主介导的整合反应。 （3）宿主整合反应经常引起相对较大的重复（数百至数千个核苷酸），而宿主序列的缺失较少。 （4）宿主连接反应通常但并非总是涉及病毒序列的丧失。 （5）很少发生复杂的事件，涉及病毒序列的重复，宿主序列的反转或从不同染色体插入序列的插入。这些实验可能对抑制剂中次优剂量的患者的治疗有影响。次优疗法可能导致异常融合。 b。在抑制剂中发展。 Y. Pommier博士正在测试由T. Burke博士开发的化合物，以抑制体外抑制HIV整合的能力（使用纯化的重组剂）；我们正在测试它们对培养细胞中病毒复制及其毒性的影响。直到最近，还不能使用结构信息来指导抑制剂的发展。然而，P. Cherepanov博士及其同事在与DNA底物和抗IN药物的复合物中获得了全长泡沫病毒（FV）的高分辨率结构。 Cherepanov博士加入了我们的合作，并将与Burke博士开发的一些更有希望的化合物一起解决复杂的FV结构。 FV活动位点与HIV-1活性位点相似，但并不相同，因此FV结构应该有用。但是，应该可以突变FV活动位点周围的区域，使其与HIV-1更相似，这应该为更有效抑制剂的设计/开发提供更好的指导。 c。重定向HIV-1 DNA的整合。重定向HIV-1 DNA的整合有可能使基因治疗更安全，因为它可能有助于解决与肠插入癌基因相关的问题。另外，该技术可用于确定基因组蛋白/结构域与染色质结合的位置。晶状体上皮衍生的生长因子（LEDGF）与HIV-1 IN相互作用，HIV-1将HIV-1 DNA整合引导到表达基因的身体。 LEDGF的C末端包含一个结合结构域，N末端结合了染色质。我们和其他人表明，用其他蛋白质代替染色质结合结构域（CBD）的LEDGF的N末端会改变HIV-1 DNA整合的特异性。最初的实验是用单个CBD进行的，或者在一种情况下是来自较大蛋白质的两个结构域。我们将进行许多其他实验：（1）我们将研究多个CBD相互作用以定义蛋白质结合染色质的特异性。最终，我们将确定全蛋白如何结合染色质。我们将从合作者C.D.艾利斯。 （2）我们到目前为止分析的所有域上结合了组蛋白尾巴上的标记。我们已经开始将技术应用于结合特定DNA序列的蛋白质，并将研究以其他方式与染色质相互作用的结构域/蛋白质。 （3）在不同组织和某些疾病状态（如癌症）中表达的基因表达不同。这意味着染色质的变化。我们刚刚从我们的合作者A. Engelman博士，Ledgf敲除小鼠那里获得，并将使用这些小鼠的细胞/组织来确定分化和转化如何影响某些良好特征良好的CBD-IBD融合的分布。 （4）我们将把技术调整到人类细胞中。 [对应于Hughes Project 2在2007年4月的现场访问艾滋病毒耐药计划报告中]