Mechanism of HIV-1 infection of urinary podocytes in HIVAN

HIVAN尿足细胞HIV-1感染机制

• 批准号: 8012356
• 负责人: Waldemar Popik
• 金额: $ 19.95万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8012356
• 关键词: AIDS-Associated Nephropathy; Adhesions; African; African American; Alleles; Apoptotic; Biological Assay; Biopsy; Blood; CCR5 gene; CD4 Antigens; CXCR4 gene; Candidate Disease Gene; Cell fusion; Confocal Microscopy; Cytoplasm; Cytoskeleton; DNA; Data; Detection; Development; Diagnosis; Disease; Dynamin; Endocytosis; Epithelial Cells; Filtration; Focal Segmental Glomerulosclerosis; Gagging; Generations; Genes; Genetic; Genetic Polymorphism; HIV; HIV-1; Human; In Vitro; Individual; Infection; Kidney; Kidney Diseases; Label; Laboratories; Lactamase; Lead; Mutation; Nested PCR; Nucleic Acids; Parietal; Pathogenesis; Pathway interactions; Patients; Play; Predisposition; Process; Proteins; RNA Splicing; Reaction; Renal glomerular disease; Reporting; Research Personnel; Reverse Transcription; Risk; Risk Assessment; Role; Sampling; Source; Specificity; Structural Protein; Testing; Time; Transcript; Tubular formation; Ultracentrifugation; Urine; Viral; Viral Genome; Viral Proteins; Virion; Virus; Visceral; Western Blotting; blood filtration; genome wide association study; in vivo; kidney cell; kidney infection; mutant; particle; podocyte; prevent; public health relevance; slit diaphragm; urinary

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.

描述（由研究者提供）：HIV-1相关肾病（HIVAN）是一种主要限于非洲裔个体的疾病。虽然其发病机制尚未完全了解，但已知HIV-1基因的表达与个体的遗传学相互作用是HIVAN所需的。全基因组研究最近将MYH 9确定为与非洲裔美国人中HIV相关局灶节段性肾小球硬化症（FSGS）发展风险相关的候选基因。虽然肾脏感染的机制尚未确定，但已在诊断为HIVAN的患者的足细胞中检测到HIV-1核酸和蛋白质。由于肾活检通常不进行HIV-1感染的患者没有肾脏疾病，这是不确定的HIV-1是否可以感染肾上皮细胞在没有艾滋病毒的患者。对于血液过滤屏障至关重要，由于HIV-1感染导致的足细胞丢失到尿液中可能会促进HIV相关的FSGS。以前的研究和我们的初步数据表明，尿液是一个可行的人类足细胞的来源。从HIVAN患者尿液中分离的足细胞可以帮助我们阐明HIVAN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CD 4、CXCR 4或CCR 5，因此病毒不太可能通过该途径进入。我们表明，HIV-1很容易进入足细胞通过一个内吞过程，导致未整合的HIV-1 DNA，不表达HIV-1结构蛋白。体内检测HIVAN足细胞中的HIV-1核酸和蛋白可能并不反映生产性复制，而是HIV-1内吞作用导致病毒基因组的有限逆转录而不整合。已显示未整合的HIV- 1 DNA的转录活性指导剪接的病毒转录物的表达和HIV-1辅助蛋白（包括Nef）的合成。Nef是HIVAN中最早和最丰富表达的病毒蛋白质之一，被认为是破坏足细胞的蛋白质。我们已经证明Nef在体外由HIV-1感染的足细胞与外泌体联合释放。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，Nef在外泌体内的传播可能会将损伤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进入足细胞导致流产感染加上分泌富含Nef的外泌体，从而使未感染的足细胞失调，导致HIVAN。为了验证这一假设，我们提出：1）通过融合试验、蛋白质印迹、共聚焦显微镜和实时PCR研究从正常供体和无HIVAN和有HIVAN的HIV+患者的尿液中分离的足细胞的HIV-1感染机制，和2）检查由HIV-1感染的足细胞分泌的外泌体和抑制Nef在促进HIVAN中的潜在作用。通过分析暴露于含Nef外泌体的未感染足细胞中细胞骨架、粘附和狭缝隔膜组分的变化以及促凋亡因子的表达，确定未感染足细胞中的特异性损伤。 公共卫生相关性：HIV相关肾病（HIV相关肾病，或HIVAN）几乎完全限于非洲裔人，超过90%的HIVAN患者是黑人。在实验室中，HIV并不容易感染肾细胞，因此本项目试图发现HIVAN是否是HIV感染肾脏的直接结果，或者这种效果是否是由于病毒的某些其他作用或身体对其的反应，从而了解病毒与特定肾细胞（称为足细胞）的相互作用这可能有助于我们开发治疗方法或早期评估非洲人后裔患艾滋病毒的风险。