Dissecting the Role of HIV-1 Tat and the Chemokine Axis in Subtype-Specificity of

剖析 HIV-1 Tat 和趋化因子轴在亚型特异性中的作用

• 批准号: 7806581
• 负责人: Vinayaka R. Prasad
• 金额: $ 58.78万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-12 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7806581
• 关键词: AIDS Dementia Complex; Address; Affect; Antibodies; Applications Grants; Astrocytes; Biological; Biological Assay; Brain; Breeding; CCL2 gene; CMV promoter; Cell Line; Cells; Cercopithecine Herpesvirus 1; Chemotaxis; Collaborations; Competence; Cysteine-Rich Domain; DNA Sequence; Data; Defect; Dementia; Development; Encephalopathies; Ensure; Evaluation; Exons; Exposure to; Flow Cytometry; Gap Junctions; Gene Expression; Gene Expression Profiling; Gene Silencing; Genes; Genetic; Genetic Polymorphism; HIV; HIV Infections; HIV encephalitis; HIV-1; Human; Hybrids; Immigration; Impaired cognition; In Vitro; Incidence; India; Individual; Infection; Infiltration; Inflammatory; Injection of therapeutic agent; Knockout Mice; Laboratory Animals; Lead; Lentivirus Vector; Leukocytes; Maps; Methods; Modeling; Molecular Cloning; Monocyte Chemoattractant Proteins; Mononuclear; Mouse Strains; Mus; Mutation; Nature; Neurologic; Neuropathogenesis; Pathogenesis; Pathway interactions; Patients; Phagocytes; Play; Point Mutation; Population; Process; Proteins; RNA Interference; Radial; Recombinants; Reporting; Retroviral Vector; Reverse Transcriptase Polymerase Chain Reaction; Role; SCID Mice; Series; Site; Small Interfering RNA; Specificity; System; Testing; Time; Tissue-Specific Gene Expression; Variant; Vertebral column; Viral Genes; Viral Genome; Virus; Water; Work; arm; base; chemokine; chemokine receptor; citrate carrier; comparative; cytokine; fitness; genome-wide; improved; knockout gene; macrophage; migration; monocyte; monocyte chemoattractant protein 1 receptor; mouse model; mutant; neuropathology; novel; research study; tat Genes; tat Protein

DESCRIPTION (provided by applicant): HIV-associated dementia (HAD) affects a significant number of HIV-infected individuals. A lower incidence/occurrence of HAD has been reported in populations with subtype C HIV-1 infections. We previously reported that subtype C HIV-1 Tat protein is defective in monocyte chemotaxis and proposed this as the basis for the reduction in HIV dementia in India. We have generated new evidence for subtype- specificity of HAD using subtype B and C isolates using the SCID HIV encephalitis (HIVE) model in mice. The current proposal includes a series of experiments to delineate the determinants of subtype-specific differences in with a special focus on Tat protein. The proposal attempts to address whether Tat alone governs the subtype differences observed, to identify the critical residues in Tat necessary for HIVE, to examine the importance of the Tat-chemokine nexus in HIVE and finally identify cellular genes specifically induced by subtype B Tat protein using differential gene expression profiling. We propose four specific aims. In Aim 1, we will examine the hypothesis that Tat gene alone is responsible for the differential ability of subtype B and C HIV-1 isolates to cause HIV dementia by introducing macrophages expressing the respective Tat genes into SCID mouse brains (in the absence of HIV infection) followed by evaluating mice for encephalopathy and cognitive dysfunction. In Aim 2, we will attempt to identify whether the dicysteine motif of Tat is the sole determinant of subtype-specificity in the HIVE model and in monocyte chemotaxis by creating point mutations in the dicysteine motif and if the results suggest otherwise, will also examine the signature residues in subtype C Tat for their importance in monocyte chemotaxis and HIVE. Importantly, we will examine the variant Tat genes isolated from the infrequently observed dementia cases among HIV-1 infected individuals in India for their role in causing HAD. In Aim 3, we will examine the role of monocyte chemoattractant protein, MCP-1 and its receptor, CCR2 by first generating gene knockout mice lacking each of these genes respectively in the SCID background followed by the intracranial injection of HIV-1 infected human macrophages. This will allow us to establish the role of chemokines in HIVE as well as dissect the role of these chemokines in the host vs. graft (via gene knockout for the murine genes and siRNA for human macrophages). Finally, we wish to identify the genes induced by HIV-1 Tat B and C proteins upon HIV-1 infection of monocytic cell line THP-1 with a view to delineate the precise pathways that lead to HAD. Proteins encoded by the genes thus identified will first be tested, using antibodies and RNA silencing in a novel in vitro chemotaxis assay we have developed to study monocyte/leukocyte migration in the context of HIV-infected macrophages. HIV-associated dementia (HAD) and other neurological complications associated with HIV-infection are on the rise. Recent reports indicate that certain strains of HIV-1 prevalent outside the US cause a lower incidence of HAD, allowing us to compare US strains to those in the above regions of the world (India). The current application provides evidence that such differences can be reproduced in small, laboratory animals and seeks to delineate the viral genes responsible for HAD.

描述（由申请人提供）：HIV 相关痴呆 (HAD) 影响大量 HIV 感染者。据报道，C 亚型 HIV-1 感染人群中 HAD 的发病率/发生率较低。我们之前报道过，C 亚型 HIV-1 Tat 蛋白在单核细胞趋化性方面存在缺陷，并提议将此作为减少印度艾滋病毒痴呆症的基础。我们通过小鼠 SCID HIV 脑炎 (HIVE) 模型，使用 B 型和 C 型分离株，获得了 HAD 亚型特异性的新证据。目前的提案包括一系列实验，以描述亚型特异性差异的决定因素，特别关注 Tat 蛋白。该提案试图解决 Tat 是否单独控制观察到的亚型差异，确定 HIVE 所需的 Tat 中的关键残基，检查 HIVE 中 Tat 趋化因子关系的重要性，并最终使用 B 亚型 Tat 蛋白特异性诱导的细胞基因差异基因表达谱。我们提出四个具体目标。在目标 1 中，我们将通过将表达各自 Tat 基因的巨噬细胞引入 SCID 小鼠大脑（在没有 HIV 的情况下）来检验这样的假设：Tat 基因单独导致 B 亚型和 C 亚型 HIV-1 分离株导致 HIV 痴呆的差异能力。感染），然后评估小鼠的脑病和认知功能障碍。在目标 2 中，我们将尝试通过在双半胱氨酸基序中创建点突变来确定 Tat 的双半胱氨酸基序是否是 HIVE 模型和单核细胞趋化性中亚型特异性的唯一决定因素，如果结果表明并非如此，我们还将检查C 亚型 Tat 中的特征残基在单核细胞趋化性和 HIVE 中的重要性。重要的是，我们将检查从印度 HIV-1 感染者中罕见的痴呆病例中分离出的 Tat 基因变异，以了解其在导致 HAD 中的作用。在目标 3 中，我们将首先在 SCID 背景中生成分别缺乏这些基因的基因敲除小鼠，然后颅内注射 HIV-1 感染的人类巨噬细胞，以检查单核细胞趋化蛋白 MCP-1 及其受体 CCR2 的作用。这将使我们能够确定趋化因子在 HIVE 中的作用，并剖析这些趋化因子在宿主与移植物中的作用（通过小鼠基因的基因敲除和人巨噬细胞的 siRNA）。最后，我们希望鉴定单核细胞系THP-1在HIV-1感染后由HIV-1 Tat B和C蛋白诱导的基因，以期描绘出导致HAD的精确途径。由如此鉴定的基因编码的蛋白质将首先在我们开发的新型体外趋化性测定中使用抗体和RNA沉默进行测试，以研究HIV感染的巨噬细胞中的单核细胞/白细胞迁移。 HIV 相关痴呆 (HAD) 和其他与 HIV 感染相关的神经系统并发症呈上升趋势。最近的报告表明，在美国以外流行的某些 HIV-1 毒株导致 HAD 的发病率较低，这使我们能够将美国毒株与世界上述地区（印度）的毒株进行比较。目前的申请提供了证据，表明这种差异可以在小型实验动物中重现，并试图找出导致 HAD 的病毒基因。