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.

过度假设认为，脑是逆转录病毒的重要储存库，因为它们能够感染长寿命的终末分化细胞，并且从这些细胞释放的病毒产物可引起免疫激活和神经元损伤 目的1：了解病毒在脑内持续存在的机制 如果有任何希望根除艾滋病毒，密切关注大脑中的病毒储存库是必要的。大脑是病毒潜伏的独特部位，因为它感染驻留的巨噬细胞/小胶质细胞和星形胶质细胞。这些细胞具有非常低的周转率，并且病毒进入和持续存在的机制与淋巴细胞非常不同，淋巴细胞是淋巴器官中被病毒感染的主要细胞类型。本实验室致力于病毒感染星形胶质细胞的机制研究。我们已经发现，虽然游离的病毒颗粒可以进入这些细胞，但与淋巴细胞的细胞间接触是感染星形胶质细胞的最有效方式。我们发现病毒通过CXCR4进入星形胶质细胞，并通过细胞之间紧密连接的形成来辅助，我们称之为“病毒突触”。我们实验室的另一个有趣的观察是，星形胶质细胞在细胞表面表达CD4，当细胞因子使它们容易受到HIV感染时。这有助于解释为什么星形胶质细胞在体内被大量感染，而这些细胞在体外很难被无细胞病毒感染。我们还发现，一旦进入，病毒可以进入作为宿主防御机制的内溶酶体途径。因此，调节这些通路的策略可能对大脑中储存库的建立产生重大影响。我们现在正在使用来自HIV感染者CSF的病毒和淋巴细胞来确认这些发现，以确定是否存在优先感染星形胶质细胞的HIV starins。目前正在编写两份手稿，详细说明这些调查结果。然而，大脑中细胞（小胶质细胞和星形胶质细胞）的周转率对于消除储库也是至关重要的，因此我们在炎症小鼠模型中发起了一项研究，以确定这些细胞的周转率是否可以在炎症状态期间改变。初步研究表明，在大脑内的特定区域，周转率加快。 目的2：探讨HIV和内源性逆转录病毒对神经元损伤的机制 尽管使用了抗逆转录病毒药物，并在外周对病毒进行了很好的控制，但艾滋病毒感染者继续出现认知障碍。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。其中一种蛋白质达特已被证明具有神经毒性。我们的实验室是最早证明其毒性潜力的实验室之一，我们现在正在研究它引起神经毒性的机制。我们已经发现，这种蛋白质可以在非常低的浓度下引起突触损伤，而不会引起神经元死亡。我们已经在人类神经元树突水平上表征了蛋白质和形态学变化，并正在进一步研究其潜在机制。使用类似的方法，我们正在研究内源性逆转录病毒K的包膜蛋白引起神经毒性的机制。我们已经将该基因克隆到表达载体中，建立了表达该蛋白的转基因系，并发现小鼠发展出类似于ALS的运动神经元疾病。通过行为学试验和组织病理学研究对小鼠进行了广泛表征。我们现在正在确定HERV-K在神经元中调节的机制，以及HERV-K是否可以从一个细胞传递到另一个细胞。 目的3：开发治疗方法，以防止病毒激活和在大脑中形成病毒储库。 我们正在产生具有HIV-Tat蛋白和HERV-K病毒的诱导表达的细胞系。这些细胞系将用于高通量筛选试验，以筛选反义分子和抑制其产生的小药理学化合物。目前正在制备病毒构建体，其将被克隆到合适的载体中用于转染细胞系。 总之，我们已经表明，脑中的星形胶质细胞是HIV的重要储存库，并且细胞与淋巴细胞的接触对于病毒进入是必要的，并且这些细胞中的溶酶体途径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，HIV蛋白达特和内源性逆转录病毒-K的env蛋白是神经毒性的，我们现在正在研究这些影响的潜在机制。最后，我们还发现HIV的达特蛋白可以使用独特的机制以不依赖于T细胞受体的方式刺激T细胞。我们现在将开发预防这些基因激活的治疗策略。