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: 7622555
• 负责人: Vinayaka R. Prasad
• 金额: $ 57.9万
• 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/7622555
• 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; Upper arm; Variant; Vertebral column; Viral Genes; Viral Genome; Virus; Water; Work; 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）：与艾滋病毒相关的痴呆（曾）影响大量的HIV感染者。据报道，在亚型C HIV-1感染的种群中，已经发生了较低的发生率/发生。我们先前报道说，亚型C HIV-1 TAT蛋白在单核细胞趋化性中有缺陷，并提出这是印度降低HIV痴呆症的基础。我们已经为使用小鼠中SCID HIV脑炎（HIVE）模型使用亚型B和C分离物的亚型B和C分离株产生了新的证据。当前的建议包括一系列实验，以描述亚型特异性差异的决定因素，并特别关注TAT蛋白。该提案试图解决观察到的亚型差异，以确定hive所必需的临界残基，以检查hive中Tat-emokine Nexus的重要性，并最终使用亚型B TAT蛋白使用差异基因表达分析来研究Tat-emokine Nexus的重要性。我们提出了四个具体目标。在AIM 1中，我们将研究以下假设：单独的TAT基因是亚型B和C HIV-1分离株的差异能力，通过将表达各自TAT基因表达的巨噬细胞引入SCID小鼠大脑中（在没有HIV感染的情况下）来引起HIV痴呆症（在没有HIV感染的情况下），然后评估小鼠的脑膜炎和Cognition ancephalopathy and Cognitive and Cognitive andcognitive andCognitive and Cognitive andcection。在AIM 2中，我们将尝试确定TAT的DycySteine主题是否是HIVE模型中亚型特异性的唯一决定因素以及单核细胞趋化性中的唯一决定因素，通过在DycySteine中产生点突变，并且结果是否暗示，是否暗示了该subtype c tat c tat tat c tat Inocycy inocax insocyte and nocaxis and nociS and syociS and nociste and nociS and symociS and nociS and syociss and hypive and nociste and hypive。重要的是，我们将研究从印度HIV-1感染的个体中不经常观察到的痴呆病例中分离出的变异基因，因为它们在引起引起的作用中的作用。在AIM 3中，我们将通过首先在SCID背景中分别产生缺乏这些基因的基因敲除小鼠，然后在SCID背景下，随后是HIV-1的HIV-1感染人类巨噬细胞在SCID背景中，我们将通过首先产生缺乏这些基因的基因基因敲除小鼠来检查单核细胞趋化蛋白及其受体CCR2的作用。这将使我们能够确定趋化因子在蜂巢中的作用，并剖析这些趋化因子在宿主和移植物中的作用（通过鼠基因的基因敲除鼠基因和人类巨噬细胞的siRNA）。最后，我们希望鉴定HIV-1 TAT B和C蛋白引起的基因在单核细胞系THP-1的HIV-1感染后，以描绘导致患有的精确途径。由抗体和RNA沉默在新型的体外趋化性测定中，我们已经开发出来研究了在HIV感染的巨噬细胞的背景下研究单核细胞/白细胞迁移的新型抗体和RNA沉默的蛋白质。与HIV感染相关的HIV相关痴呆症（HAD）和其他与HIV感染相关的神经系统并发症正在上升。最近的报道表明，美国以外的某些HIV-1菌株导致较低的发生率，使我们能够将美国的菌株与世界上述地区（印度）（印度）进行比较。当前的应用提供了证据表明，这种差异可以在小型实验室动物中复制，并试图描绘导致患者的病毒基因。