Effects of Exosomes Derived from HIV-1 Infected Cells on Viral Spread

说明（由申请人提供）：截至2011年，联合国艾滋病规划署关于全球艾滋病流行的报告表明，有3 400万人感染了艾滋病毒。从区域来看，美国和加拿大的感染人数为140万，每年记录的新病例为51,000例。此外，在美国，艾滋病相关原因每年夺去近2万人的生命，尽管使用了高效抗逆转录病毒治疗，但仍有高达47%的患者被诊断出与艾滋病相关的神经认知障碍。近年来，外泌体作为细胞间通讯载体的概念为宿主-病原体相互作用和病毒传播的研究开辟了新的途径。关于病毒感染的另一个新兴领域是RNA干扰途径，该途径产生mirna，引导效应复合物到达mRNA或DNA中的互补序列，从而抑制基因表达。此外，我们的实验室以及其他实验室已经确定了长期感染中整合原病毒的TAR元件衍生的mirna。虽然HIV-1病毒mirna的概念仍然存在争议，但实验室得出的结论是HIV-1不编码mirna，研究的是短期感染，而不是生物学上更相关的长期感染。此外，我们的初始数据显示来自HIV-1 TAR的病毒mirna被包装并通过外泌体从感染细胞中分泌。我们的长期目标是了解来自HIV-1感染细胞的外泌体在调节宿主-病毒相互作用中的作用。本研究的主要目的是阐明HIV改变的外泌体如何影响未感染的中枢神经系统细胞，并协助病毒传播的机制。我们假设感染细胞外泌体中的病毒mirna通过影响调节信号转导和基因表达以及病毒传播来改变受体细胞。我们假设的基本原理包括我们的数据显示，培养上清液和患者血清中的病毒mirna表明mirna处于受保护的环境中。此外，我们已经证明来自感染细胞的外泌体可以对幼稚的旁观者细胞施加功能影响，使它们更容易受到HIV感染。然而，我们目前对hiv改变的外泌体功能的理解是有限的，导致外泌体机制在中枢神经系统中导致病毒发病的知识差距。为了验证我们的假设，我们提出了以下目标：1)检查感染原代细胞中外泌体操作的改变组成和机制，并验证外泌体在中枢神经系统来源的受体细胞上的功能2)使用人源化动物测试外泌体对人脑MDMs中HIV-1复制的影响。这些研究的预期结果包括阐明外泌体组成的改变和病毒miRNA对中枢神经系统初始受体细胞信号转导、基因表达和病毒易感性增加的影响。我们的体内实验，结合外泌体抑制剂治疗后外泌体生物发生减少的验证，将建立循环外泌体与中枢神经系统感染相关表型之间的强相关性。

DESCRIPTION (provided by applicant): As of 2011, the UNAIDS report on the global AIDS epidemic indicated that 34 million people are living with HIV. Regionally, the number of infected individuals in the USA and Canada is 1.4 million with 51,000 new cases being documented every year. Furthermore, AIDS-related causes claim the lives of nearly 20,000 people in the USA annually and HIV-associated neurocognitive disorders are diagnosed in up to 47 percent of patients despite the use of highly active antiretroviral therapy. Recently, the concept of exosomes acting as vehicles of intercellular communication has opened up new avenues of study regarding host-pathogen interactions and viral spread. Another emerging field regarding viral infections is that of the RNA interference pathway which generates miRNAs that guide effector complexes to complimentary sequences in mRNA or DNA, thereby suppressing gene expression. Moreover, our lab, as well as others, has identified miRNAs derived from the TAR element of integrated provirus in long-term infections. While the concept of HIV-1 viral miRNAs remains controversial, labs concluding that HIV-1 doesn't encode miRNAs have examined short-term infections as opposed to the more biologically relevant long-term infections. Furthermore, our initial data shows viral miRNAs derived from HIV-1 TAR are packaged and secreted from infected cells through exosomes. Our long term goal is to understand the role played by exosomes from HIV-1 infected cells in regulating host-virus interactions. The primary objective of the current proposal is to elucidate the mechanisms by which HIV altered exosomes effect uninfected cells of CNS origins, and assist in viral spread. We hypothesize that viral miRNAs within exosomes of infected cells alter recipient cells by impacting regulation signal transduction and gene expression, as well as viral spread. The rationale for our hypothesis includes our data showing viral miRNAs in culture supernatants and patient sera suggesting the miRNAs are in a protected environment. Moreover, we've shown that exosomes from infected cells can exert functional influences on naive bystander cells and make them more susceptible to HIV infection. However, our current understanding of the functionality of HIV-altered exosomes is limited, resulting in a gap of knowledge regarding exosomal mechanisms that contribute to viral pathogenesis in the CNS. To test our hypothesis we propose the following aims: 1) Examine the altered composition and mechanisms of exosome manipulation in infected primary cells, and validate the functionality of exosomes on recipient cells of CNS origins 2) Test the effects of exosomes on HIV-1 replication in human MDMs within the brain using humanized animals. The expected outcomes of these studies includes elucidation of altered exosome composition and viral miRNA influences on signal transduction, gene expression and increased viral susceptibility of naive recipient cells of the CNS. Our in vivo experiments, in combination with validation of decreased exosome biogenesis following exosome inhibitor treatment, will establish a strong correlation between circulating exosomes and infection-associated phenotypes in the CNS.