Mechanism of HIV-1 infection of urinary podocytes in HIVAN

描述(由研究人员提供)：HIV-1相关性肾病(HIVAN)是一种主要局限于非洲裔个人的疾病。尽管其发病机制尚不完全清楚，但已知HIV-1基因的表达与个体的遗传学相互作用，是HIVAN所必需的。全基因组研究最近确定Myh9是一个候选基因，与非裔美国人发生HIV相关的局灶性节段性肾小球硬化(FSGS)的风险有关。虽然肾脏感染的机制尚不清楚，但已在确诊为HIVAN的患者的足细胞中检测到HIV-1核酸和蛋白质。由于通常不会对没有肾脏疾病的HIV-1感染患者进行肾脏活检，因此尚不确定HIV-1是否会感染没有HIVAN的患者的肾脏上皮细胞。对血液滤过屏障至关重要的足细胞，由于HIV-1感染而丢失到尿液中，可能会促进HIV相关的FSGS。先前的研究和我们的初步数据表明，尿液是人类足细胞存活的一个来源。从HIVAN患者尿液中分离足细胞可以帮助我们阐明HIVAN的发病机制，包括导致他们感染的机制(S)。足细胞缺乏经典的HIV-1进入受体CD4、CXCR4或CCR5，因此病毒不太可能通过这一途径进入。我们发现，HIV-1很容易通过内吞过程进入足细胞，导致不表达HIV-1结构蛋白的未整合的HIV-1DNA。在体内检测HIVAN足细胞中的HIV-1核酸和蛋白质可能不能反映出生产性复制，而是HIV-1的内吞作用导致病毒基因组的有限反转录，而不是整合。未整合的HIV-1 DNA的转录活性已被证明指导剪接病毒转录本的表达和包括Nef在内的HIV-1辅助蛋白的合成。NEF是HIVAN中最早和最丰富表达的损伤足细胞的病毒蛋白之一。我们已经证明，Nef在体外是由HIV-1感染的足细胞与外切体结合释放的。在HIV-1颗粒中也可以发现Nef，然而，只有少数HIV-1颗粒逃逸到细胞质中，在那里它们可以在去涂层的过程中释放Nef。因此，Nef在外体内的传播可能会将损害扩散到未感染的足细胞，而不会进行生产性复制。我们认为，HIV-1进入足细胞会导致流产感染，以及富含Nef的外体分泌，进而导致未感染的足细胞失调，导致HIVAN。为了验证这一假说，我们建议1)通过融合实验、Western blotting、共聚焦显微镜和实时荧光PCR来研究从正常捐赠者和HIV+患者尿液中分离出的HIV-1感染足细胞的机制，以及2)通过分析细胞骨架、黏附和裂隙横隔膜成分以及非感染足细胞中促凋亡因子的表达的变化，研究由HIV-1感染的足细胞分泌的外切体在促进未感染足细胞HIVAN特异性损伤中的潜在贡献。 公共卫生相关性：艾滋病毒相关肾病(HIV相关肾病，简称HIVAN)几乎完全局限于非洲人后裔，90%以上的HIVAN患者是黑人。在实验室里，HIV不容易感染肾脏细胞，所以这个项目试图发现HIVAN是HIV感染肾脏的直接结果，还是由于病毒的其他作用或身体对它的反应所致。了解病毒与称为足细胞的特定肾脏细胞的相互作用，可能有助于我们开发治疗方法或早期评估非洲裔人患HIVAN的风险。

DESCRIPTION (provided by investigator): HIV-1 associated nephropathy (HIVAN) is a disease largely limited to individuals of African descent. Although its pathogenesis is not completely understood, it is known that expression of HIV-1 genes, interacting with the individual's genetics, is required for HIVAN. Genome-wide studies recently identified MYH9 as a candidate gene associated with the risk for development of HIV-associated focal segmental glomerulosclerosis (FSGS) in African Americans. Although the mechanism of kidney infection remains undetermined, HIV-1 nucleic acids and proteins have been detected in podocytes of patients diagnosed with HIVAN. Since renal biopsies are not usually performed on HIV-1 infected patients without kidney disease, it is uncertain whether HIV-1 can infect renal epithelial cells in patients without HIVAN. Critical to the blood filtration barrier, podocytes lost into urine as a result of HIV-1 infection may promote HIV-associated FSGS. Previous studies and our preliminary data show that urine is a source of viable human podocytes. Podocytes isolated from the urine of HIVAN patients can help us clarify HIVAN pathogenesis including the mechanism(s) leading to their infection. Podocytes lack classical HIV-1 entry receptors CD4, CXCR4, or CCR5 so virus entry through this pathway is unlikely. We show that HIV-1 readily enters podocytes through an endocytotic process leading to unintegrated HIV-1 DNA that does not express HIV-1 structural proteins. In vivo detection of HIV-1 nucleic acids and proteins in HIVAN podocytes may not reflect productive replication but rather that HIV-1 endocytosis leads to a limited reverse transcription of the viral genome without integration. Transcriptional activity of unintegrated HIV- 1 DNA has been shown to direct the expression of spliced viral transcripts and synthesis of HIV-1 accessory proteins, including Nef. Nef is one of the earliest and most abundantly expressed viral proteins proposed to damage podocytes in HIVAN. We have shown that Nef is released in vitro by HIV-1 infected podocytes in association with exosomes. Nef can also be found in HIV-1 particles, however, only a few HIV-1 particles escape into the cytoplasm where they can release Nef during uncoating. Thus, dissemination of Nef within exosomes could spread damage to uninfected podocytes without productive replication. We propose that HIV-1 entry into podocytes results in an abortive infection plus the secretion of exosomes enriched in Nef that in turn dysregulate uninfected podocytes leading to HIVAN. To test this hypothesis, we propose 1) to investigate the mechanism of HIV-1 infection of podocytes isolated from the urine of normal donors and HIV+ patients without and with HIVAN via fusion assay, Western blotting, confocal microscopy and real-time PCR, and 2) to examine the potential contribution of exosomes secreted by HIV-1 infected podocytes and encapsidating Nef in the promotion of HIVAN-specific damage in uninfected podocytes via analysis of changes in the cytoskeleton, adhesion and slit diaphragm components as well as expression of pro-apoptotic factors in uninfected podocytes exposed to Nef-containing exosomes. PUBLIC HEALTH RELEVANCE: HIV-related kidney disease (HIV associated nephropathy, or HIVAN) is almost entirely limited to people of African descent, with over 90% of HIVAN patients being black. In the laboratory, HIV does not easily infect kidney cells, so this project seeks to discover if HIVAN is the direct result of HIV infecting the kidneys, or if the effect is due to some other action of the virus or the body's reaction to it. Understanding the interaction of the virus with specific kidney cells, called podocytes, may help us develop treatments or early assessments of the risk for developing HIVAN in people of African descent.