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

描述(由申请人提供)：艾滋病毒相关性痴呆症(HAD)影响着相当数量的艾滋病毒感染者。据报道，在感染C亚型HIV-1的人群中，HAD的发病率/发生率较低。我们之前报道了C亚型HIV-1 Tat蛋白在单核细胞趋化能力方面存在缺陷，并建议将其作为减少印度HIV痴呆的基础。我们利用小鼠的SCID HIV脑炎(HIVE)模型，使用B和C亚型分离株，为HAD的亚型特异性提供了新的证据。目前的建议包括一系列实验，以描绘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TAT是否单独控制观察到的亚型差异，确定TAT中对HIVE必需的关键残基，检查TAT-趋化因子关系在HIVE中的重要性，并最终利用差异基因表达谱识别由B亚型TAT蛋白特异性诱导的细胞基因。我们提出了四个具体目标。在目标1中，我们将通过将表达各自TAT基因的巨噬细胞导入SCID小鼠的大脑(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基因单独导致B型和C型HIV-1分离株导致HIV痴呆的区别能力的假设。在目标2中，我们将试图通过在二半胱氨酸基序中产生点突变来确定TAT的二半胱氨酸基序是否是HIVE模型和单核细胞趋化中亚型特异性的唯一决定因素，如果结果不是这样，我们还将检查C亚型TAT中的签名残基在单核细胞趋化和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症病例中分离出的变异TAT基因，以寻找它们在导致HAD中的作用。在目标3中，我们将首先在SCID背景下产生分别缺失单核细胞趋化蛋白MCP-1及其受体CCR2的基因敲除小鼠，然后将HIV-1感染的人巨噬细胞脑内注射。这将使我们能够确定趋化因子在蜂箱中的作用，并剖析这些趋化因子在宿主和移植物中的作用(通过基因敲除小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蛋白在单核细胞系THP-1感染HIV-1过程中诱导的基因，以期描绘导致HAD的确切途径。我们开发了一种新的体外趋化试验来研究HIV感染的巨噬细胞中单核细胞/白细胞的迁移，首先将使用抗体和RNA沉默来测试由这样识别的基因编码的蛋白质。艾滋病毒相关性痴呆症(HAD)和其他与艾滋病毒感染相关的神经系统并发症正在增加。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的毒株与世界上上述地区(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室动物身上复制，并试图描绘导致HAD的病毒基因。

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.