Mechanism of HIV-1 infection of urinary podocytes in HIVAN

描述（由研究者提供）：HIV-1相关肾病（HIVAN）是一种主要局限于非洲人后裔的疾病。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相互作用，是HIVAN的必要条件。最近的全基因组研究发现，MYH9是与非裔美国人hiv相关局灶节段性肾小球硬化（FSGS）发展风险相关的候选基因。尽管肾脏感染的机制尚未确定，但在诊断为hiv - van的患者足细胞中检测到HIV-1核酸和蛋白质。由于没有肾脏疾病的HIV-1感染患者通常不进行肾脏活检，因此不确定HIV-1是否可以感染没有hiv - van的患者的肾上皮细胞。足细胞是血液过滤屏障的关键，由于HIV-1感染而丢失到尿液中的足细胞可能会促进hiv相关的FSGS。先前的研究和我们的初步数据表明，尿液是人类足细胞的一个来源。从hiv患者尿液中分离的足细胞可以帮助我们阐明hiv的发病机制，包括导致其感染的机制。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HIV-1 DNA。HIV-1核酸和蛋白质在HIVAN足细胞中的体内检测可能不能反映有效复制，而是HIV-1内吞作用导致病毒基因组的有限逆转录而没有整合。未整合HIV-1 DNA的转录活性已被证明可以指导剪接病毒转录物的表达和HIV-1附属蛋白（包括Nef）的合成。Nef是最早和最丰富表达的破坏HIVAN足细胞的病毒蛋白之一。我们已经证明，Nef与外泌体一起在体外被HIV-1感染的足细胞释放。Nef也可以在HIV-1颗粒中发现，然而，只有少数HIV-1颗粒逃逸到细胞质中，在那里它们可以在脱壳过程中释放Nef。因此，外泌体内Nef的传播可以将损伤传播到未感染的足细胞而不进行有效复制。我们认为，HIV-1进入足细胞导致感染流产，加上富含Nef的外泌体的分泌，进而失调未感染的足细胞，导致HIV-1感染。为了验证这一假设，我们提出：1)通过融合实验、Western blotting、共聚焦显微镜和实时PCR，研究从正常供体和HIV+患者的尿液中分离的足细胞感染HIV-1的机制，以及2)通过分析细胞骨架的变化，检测HIV-1感染足细胞分泌的外泌体和封装Nef在促进未感染足细胞中HIV-1特异性损伤中的潜在作用。暴露于含nef外泌体的未感染足细胞中粘附和狭缝隔膜成分以及促凋亡因子的表达。

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.