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

描述（由申请人提供）：HIV相关性痴呆（HAD）影响大量HIV感染者。据报道，在C亚型HIV-1感染人群中HAD的发病率/发生率较低。我们以前报道过C亚型HIV-1达特蛋白在单核细胞趋化性方面存在缺陷，并提出这是印度HIV痴呆症减少的基础。我们在小鼠中使用SCID HIV脑炎（HIVE）模型，使用亚型B和C分离株，获得了HAD亚型特异性的新证据。目前的建议包括一系列的实验，以描绘亚型特异性差异的决定因素，特别是对达特蛋白。该提案试图解决是否达特单独管理亚型差异观察，以确定关键的残基达特必要的HIVE，检查的重要性，在HIVE的塔特-趋化因子的关系，并最终确定细胞基因特异性诱导亚型B达特蛋白使用差异基因表达谱。我们提出了四个具体目标。在目的1中，我们将通过将表达相应达特基因的巨噬细胞引入SCID小鼠脑（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，来检验达特基因单独负责亚型B和C HIV-1分离株引起HIV痴呆的差异能力的假设。在目标2中，我们将尝试通过在二半胱氨酸基序中产生点突变来确定达特的二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，如果结果表明不是这样，我们还将检查亚型C达特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，我们将研究变异达特基因分离的罕见观察到的痴呆症病例中的HIV-1感染者在印度的作用，导致HAD。在目的3中，我们将通过首先在SCID背景下分别产生缺乏这些基因中的每一个的基因敲除小鼠，然后颅内注射HIV-1感染的人巨噬细胞来检查单核细胞趋化蛋白MCP-1及其受体CCR 2的作用。这将使我们能够确定趋化因子在HIVE中的作用，以及剖析这些趋化因子在宿主与移植物中的作用（通过鼠基因的基因敲除和人巨噬细胞的siRNA）。最后，我们希望确定由HIV-1达特B和C蛋白诱导的基因，HIV-1感染单核细胞系THP-1，以描绘导致HAD的精确途径。由此确定的基因编码的蛋白质将首先进行测试，使用抗体和RNA沉默在一个新的体外趋化性测定，我们已经开发了研究单核细胞/白细胞迁移的背景下，HIV感染的巨噬细胞。艾滋病毒相关性痴呆（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，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。本申请提供了这样的差异可以在小的实验室动物中重现的证据，并试图描绘负责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.