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

剖析 HIV-1 Tat 的作用

• 批准号: 8249779
• 负责人: Vinayaka R. Prasad
• 金额: $ 57.39万
• 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 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8249779
• 关键词: AIDS Dementia Complex; Address; Affect; Antibodies; Applications Grants; Astrocytes; Biological; Biological Assay; Brain; Breeding; CCL2 gene; CMV promoter; Cell Line; Cells; 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）影响了大量hiv感染者。据报道，在C型HIV-1感染人群中，HAD的发病率较低。我们之前报道过C型HIV-1 Tat蛋白在单核细胞趋化性方面存在缺陷，并提出这是印度HIV痴呆减少的基础。我们在小鼠SCID HIV脑炎（HIVE）模型中使用B和C亚型分离物获得了HAD亚型特异性的新证据。目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，特别关注Tat蛋白。该提案试图解决Tat是否单独控制所观察到的亚型差异，确定HIVE所需的Tat中的关键残基，检查HIVE中Tat-趋化因子联系的重要性，并最终使用差异基因表达谱确定B型Tat蛋白特异性诱导的细胞基因。我们提出了四个具体目标。在Aim 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感染的情况下），然后评估小鼠的脑病和认知功能障碍，来检验Tat基因单独负责亚型B和C HIV-1分离物引起HIV痴呆的差异能力的假设。在Aim 2中，我们将通过在二半胱氨酸基序中创建点突变来尝试确定Tat的二半胱氨酸基序是否是HIVE模型中亚型特异性和单核细胞趋化性的唯一决定因素，如果结果相反，我们还将检查C型Tat的特征残基在单核细胞趋化性和HIVE中的重要性。重要的是，我们将研究从印度HIV-1感染者中罕见观察到的痴呆病例中分离出的Tat变异基因在HAD中的作用。在Aim 3中，我们将通过首先在SCID背景下分别产生缺乏这些基因的基因敲除小鼠，然后在颅内注射HIV-1感染的人巨噬细胞，来研究单核细胞趋化蛋白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和移植物中的作用（通过敲除小鼠基因和敲除人类巨噬细胞的siRNA）。最后，我们希望鉴定HIV-1 Tat B和C蛋白在单核细胞系THP-1感染HIV-1时诱导的基因，以期描绘导致HAD的精确途径。我们开发了一种新的体外趋化试验，用于研究hiv感染巨噬细胞背景下单核细胞/白细胞的迁移。通过抗体和RNA沉默，我们将首先测试由这些基因编码的蛋白质。艾滋病毒相关痴呆（HAD）和其他与艾滋病毒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上述地区（印度）的毒株进行比较。目前的应用提供了证据，证明这种差异可以在小型实验室动物中重现，并试图描述导致HAD的病毒基因。

项目成果

Methods to study monocyte migration induced by HIV-infected cells.

研究 HIV 感染细胞诱导的单核细胞迁移的方法。

• DOI: 10.1007/978-1-59745-170-3_20
• 发表时间: 2009
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Rao,VasudevR;Eugenin,EliseoA;Berman,JoanW;Prasad,VinayakaR
• 通讯作者: Prasad,VinayakaR