Neuropathogenesis of Retroviral Infections

逆转录病毒感染的神经发病机制

• 批准号: 9157565
• 负责人: Avindra Nath
• 金额: $ 424.5万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9157565
• 关键词: Anti-Retroviral Agents; Area; Astrocytes; Attention; Biological Assay; Brain; CXCR4 gene; Cell Line; Cell surface; Cells; Cessation of life; Chromosomes; Dendrites; Endogenous Retroviruses; Gene Activation; Genetic Vectors; Goals; HERVs; HIV; Host Defense Mechanism; Human; Impaired cognition; In Vitro; Individual; Infection; Inflammation; Injury; Laboratories; Lead; Life; Lymphocyte; Lymphoid; Manuscripts; Microglia; Motor Neuron Disease; Mus; Neurologic; Neuronal Injury; Neurons; Neuropathogenesis; Organ; Pathway interactions; Patients; Production; Proteins; Retroviridae; Site; Synapses; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; Tight Junctions; Transgenic Organisms; Viral; Viral Proteins; Virus; Virus Diseases; Virus Latency; cell type; cytokine; env Gene Products; expression vector; gene cloning; immune activation; in vivo; interest; macrophage; mouse model; neurotoxic; neurotoxicity; particle; prevent; screening; small molecule; tat Protein; trafficking

The over arching hypothesis is that the brain is an important reservoir for retroviruses because of their ability to infect long lived terminally differentiated cells and viral products released from these cells can cause immune activation and neuronal injury Aim 1: To understand the mechanism of viral persistence in brain If there is any hope to eradicate HIV, close attention to the viral reservoirs in the brain is necessary. The brain is a unique site of viral latency since it infects resident macrophages/microglia and astrocytes. These cells have very low turnover rate, and the mechanism of viral entry and persistence is very different than that of lymphocytes which are the major cell type infected by the virus in the lymphoid organs. Our laboratory has focused its efforts on studying the mechanism of viral infection of astrocytes. We have found that while free viral particles can enter these cells, cell to cell contact with lymphocytes is the most efficient way to infect astrocytes. We have discovered that the virus enters astrocytes by using CXCR4 and is aided by formation of tight junctions between the cells which we have termed, "viral synapses".Another interesting observation made in our laboratory is that astrocytes express CD4 on the cell surface when eposed to cytokines which makes them vulnerable to HIV infection. This helps explain why astrocytes in vivo are infected in large numbers yet these cells have been very difficult to infect in vitro with cell free virus. We have also found that upon entry, the virus can enter the endolysosomal pathway which acts as a host defense mechanism. Hence strategies than modulate these pathways could have a significant effect on the establishment of a reservoir in the brain. We are now confirming these findings using virus and lymphocytes from CSF of HIV infected individuals to determine if there are starins of HIV that preferentially infect astrocytes. Two manuscripts are currently being prepared that detail these findings. However the turnover rate of the cells in the brain (microglia and astrocytes) is also critical to the eradication of the reservoir, hence we have initated a study in a mouse model of inflammation to determine if the turnover rate of these cells may be altered during the state of inflammation. preliminary studies suggest that the turnover rate is accelerated in specific areas within the brain. Aim 2: To investigate the mechanism of neuronal injury by HIV and endogenous retroviruses Despite the use of antiretroviral agents and excellent control of the virus in the periphery, HIV infected patients continue to develop cognitive impairment. Currently available antiretroviral agents have no effect on the production of early viral proteins once the virus has integrated into the chromosome. One of these proteins, Tat, has been shown to be neurotoxic. Our laboratory was one of the first to demonstrate its toxic potential and we are now investigating the mechanisms by which it causes neurotoxicity. We have found that the protein can cause synaptic injury at very low concentrations without causing neuronal death. We have characterized the proteins and the morphological changes at the level of the dendrites in human neurons and are further investigating the underlying mechanisms. Using a similar approach we are investigating the mechanisms by which the envelop protein of an endogenous retrovirus-K causes neurotoxicity. We have cloned the gene into an expression vector, created a transgenic line that expresses the protein and have found that the mice develop a motor neuron disease simialr to ALS. The mice have been extensively characterized by behvioral testing and histopathological studies. We are now determinign the mechanism by which HERV-K is regulated in neurons and if HERV-K can be transmitted from one cell to another. Aim 3: To develop therapeutic approaches to prevent viral activation and formation of viral reservoirs in the brain. We are generating cell lines with inducible expression of HIV-Tat protein and the HERV-K virus. These cell lines will be used in high through put screening assays to screen for anti-sense molecules and for small pharmacological compounds that suppress their production. Currently viral constructs are being prepared, which will be cloned into appropriate vectors for transfection of cell lines. In summary, we have shown that astrocytes in the brain are an important reservoir for HIV and that cell to cell contact with lymphocytes is necessary for viral entry and the lysosomal pathway in these cells regulates the intracellular trafficking of the virus and its ultimate ability to successfully infect these cells. Further, we have shown that the HIV protein Tat and the env protein of endogenous retrovirus-K are neurotoxic and we are now studying the underlying mechanisms involved in these effects. Finally, we have also discovered that the Tat protein of HIV can stimulate T cells in a T cell receptor independent manner using a unique mechanism. We will now develop therapeutic strategies for preventing the activation of these genes.

过度拱形假设是大脑是逆转录病毒的重要储层，因为它们能够感染长期终极分化的细胞和从这些细胞释放的病毒产物的能力会引起免疫激活和神经元损伤 目标1：了解大脑病毒持续性的机制 如果有任何希望消除艾滋病毒的希望，则需要密切注意大脑中的病毒储存库。大脑是病毒潜伏期的独特部位，因为它感染了常驻巨噬细胞/小胶质细胞和星形胶质细胞。这些细胞的周转率非常低，病毒入口和持久性的机制与淋巴细胞的机制大不相同，淋巴细胞是由病毒感染的主要细胞类型。我们的实验室将其努力集中在研究星形胶质细胞病毒感染机制上。我们发现，尽管游离病毒颗粒可以进入这些细胞，但细胞与淋巴细胞接触的细胞是感染星形胶质细胞的最有效方法。我们已经发现，该病毒通过使用CXCR4进入星形胶质细胞，并通过我们称为“病毒突触”的细胞之间形成紧密连接的帮助，在我们的实验室中进行的其他有趣的观察是，当使它们易于细胞因子时，星形胶质细胞在细胞表面上表达CD4，这使得它们使其对HIV感染变得脆弱。这有助于解释为什么体内星形胶质细胞被大量感染，但这些细胞在体外很难用无细胞病毒感染。我们还发现，进入后，该病毒可以进入宿主防御机制的内溶性途径。因此，策略比调节这些途径可能会对大脑中的水库建立产生重大影响。我们现在正在使用来自HIV感染个体CSF的病毒和淋巴细胞来证实这些发现，以确定是否有优先感染星形胶质细胞的HIV的Starins。目前正在准备两个详细说明这些发现的手稿。但是，大脑中细胞的周转率（小胶质细胞和星形胶质细胞）对于消除储层也至关重要，因此我们已经在炎症的小鼠模型中启动了一项研究，以确定在炎症状态期间这些细胞的周转率是否可能改变。初步研究表明，在大脑内的特定区域中，周转率加速了。 目标2：研究HIV和内源性逆转录病毒的神经元损伤机制 尽管使用抗逆转录病毒药物并在周围病毒中极好控制病毒，但感染HIV的患者仍会继续发展认知障碍。一旦病毒整合到染色体中，目前可用的抗逆转录病毒药物对早期病毒蛋白的产生没有影响。这些蛋白质之一TAT已被证明是神经毒性的。我们的实验室是最早证明其有毒潜力的实验室之一，我们现在正在研究引起神经毒性的机制。我们发现该蛋白质会在非常低浓度的情况下引起突触损伤而不会导致神经元死亡。我们已经表征了人类神经元中树突水平上的蛋白质和形态变化，并正在进一步研究基本机制。使用类似的方法，我们正在研究内源性逆转录病毒-K的包蛋白引起神经毒性的机制。我们将基因克隆到表达载体中，创建了一种表达蛋白质的转基因线，并发现小鼠与ALS形成了运动神经元疾病相似。小鼠通过概念测试和组织病理学研究的广泛特征。现在，我们确定了在神经元中调节HERV-K的机制，并且是否可以将HERV-K从一个细胞传输到另一个细胞。 目标3：开发治疗方法，以防止大脑中病毒式储藏的病毒激活和形成。 我们正在生成具有HIV-TAT蛋白和HERV-K病毒的诱导表达的细胞系。这些细胞系将通过放置筛选测定法以筛选抗敏感性分子以及抑制其产生的小药理学化合物。目前正在准备病毒构建体，将其克隆到适当的载体中以转染细胞系。 总而言之，我们已经表明，大脑中的星形胶质细胞是HIV的重要储备，并且细胞与细胞与淋巴细胞的接触对于病毒的进入是必要的，并且这些细胞中的溶酶体途径是调节病毒的细胞内运输及其成功感染这些细胞的最终能力。此外，我们已经表明，内源性逆转录病毒-K的HIV蛋白TAT和ENV蛋白是神经毒性的，我们现在正在研究这些作用所涉及的潜在机制。最后，我们还发现，HIV的TAT蛋白可以使用独特的机制以独立的T细胞受体方式刺激T细胞。现在，我们将制定预防这些基因激活的治疗策略。