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

描述(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显示，有3400万人携带艾滋病毒。在区域范围内，美国和加拿大的感染者人数为140万人，每年新增5.1万例病例。此外，在美国，与艾滋病相关的原因每年夺走近2万人的生命，尽管使用了高效的抗逆转录病毒疗法，但仍有高达47%的患者被诊断出与艾滋病毒相关的神经认知障碍。最近，外切体作为细胞间通讯载体的概念为研究宿主与病原体的相互作用和病毒传播开辟了新的途径。另一个关于病毒感染的新兴领域是RNA干扰途径，它产生miRNAs，引导效应器复合体在mRNA或DNA中互补序列，从而抑制基因表达。此外，我们的实验室和其他实验室一样，已经在长期感染中发现了来自整合前病毒的焦油成分的miRNAs。虽然HIV-1病毒miRNAs的概念仍然存在争议，但得出HIV-1病毒不编码miRNAs的实验室已经检查了短期感染，而不是更具生物学意义的长期感染。此外，我们的初步数据显示，来自HIV-1TAR的病毒miRNAs是通过外切体从感染细胞包装和分泌的。我们的长期目标是了解HIV-1感染细胞的外切体在调节宿主-病毒相互作用中所起的作用。目前这项提议的主要目的是阐明HIV改变的外切体影响源自CNS的未感染细胞的机制，并帮助病毒传播。我们假设，感染细胞外体内的病毒miRNAs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，在培养上清液和患者血清中存在病毒miRNAs，这表明miRNAs处于受保护的环境中。此外，我们已经证明，来自受感染细胞的外切体可以对幼稚的旁观者细胞产生功能影响，使它们更容易受到艾滋病毒感染。然而，我们目前对HIV改变的外切体的功能的了解是有限的，导致对促进中枢神经系统病毒致病的外切体机制的了解存在差距。为了验证我们的假设，我们提出了以下目标：1)检查感染的原代细胞中外切体操纵的改变的组成和机制，并验证外切体在中枢神经系统起源的受体细胞上的功能2)使用人源化动物测试外切体对人类MDM中HIV-1复制的影响。这些研究的预期结果包括阐明外切体组成的改变以及病毒miRNA对中枢神经系统幼稚受体细胞的信号转导、基因表达和病毒易感性的影响。我们的体内实验，结合外切体抑制剂治疗后外切体生物生成减少的验证，将建立循环外切体与中枢神经系统感染相关表型之间的强烈相关性。

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.