BK Virus Receptor and polyomavirus nephropathy

描述(申请人提供)：近年来，由BK病毒(BKV)引起的肾炎已成为肾移植后的严重问题。BKV是一种无膜双链DNA多瘤病毒。迫切需要更好地确定BKV进入其靶细胞的分子机制，并开发有效的BKN治疗策略。本研究的目的是确定BKV受体的蛋白成分，并开发能够减轻BKV进入人肾近端小管上皮细胞(HRPTEC)的药物，HRPTEC被认为是BKV的主要天然靶点之一。BKV受体的身份尚不清楚。目前尚无预防BKV肾炎的特效药。目前，治疗BKV肾炎的唯一有效方法似乎是减少/改变免疫抑制剂，这可能会增加固有的排斥反应风险。在我们的初步研究中，我们已经证实空泡内吞作用是BKV感染HRPTEC的关键，并揭示了HRPTEC中BKV细胞内转运途径的几个重要步骤。此外，我们的数据表明，内皮素-1与内毒素B受体(ETRB)结合的拮抗剂可以预防BKV感染HRPTEC。内皮素(Endothelins，ET)是G蛋白偶联受体的激动剂，是肾脏疾病进展的重要介质。我们的工作假设是，在HRPTEC中，BKV与ETRB的结合是BKV进入HRPTEC的关键步骤，ETRB拮抗剂可以有效地治疗BKV肾炎。特定目标1将检验N-连接糖蛋白ETBR在HRPTEC中作为BKV颗粒受体的假设。为了确定ETRB是HRPTEC中BKV颗粒的结合分子，将采用siRNA介导的ETRB基因沉默的方法。我们还将进行实验，以确定ETRB和BKV颗粒之间的直接联系，并评估ETRB存在对BKV感染的必要性。BKV感染的检测将使用以下标志物：免疫荧光法检测感染细胞的百分比，Western印迹分析检测BKV大T抗原表达的细胞水平，以及实时荧光聚合酶链式反应(Real-Time PCR)检测病毒载量。拟议的研究将为BKN的治疗干预提供新的策略，最终提高肾移植后移植物的长期存活率。 公共卫生相关性：近年来，由无包膜双链脱氧核糖核酸多瘤病毒BK病毒(BKV)引起的肾炎已成为肾移植后的严重问题。多瘤病毒肾病，也称为BK病毒肾病(BKN)，影响1%至10%的肾移植受者，并正在成为一种日益严重的威胁。由于在患有BKN的肾移植受者中，由于BKN造成的移植物丢失从10%到80%以上，现在迫切需要更好地确定BKV进入其靶细胞的分子机制，并开发有效的BKN治疗策略。BKV受体的特性尚不清楚，目前还没有预防BKV肾炎的特定药理药物。这一建议将揭示BKV受体的蛋白成分，并为BKN治疗干预的新策略提供基础，最终提高肾移植后移植物的长期存活率。

DESCRIPTION (provided by applicant): In recent years, nephritis induced by BK virus (BKV), a non-enveloped double-stranded DNA polyomavirus, has become a severe problem after renal transplantation. There is a critical need to better define the molecular mechanisms of BKV entry into its target cells and to develop efficient treatment strategies for BKN. An objective of the proposed study is to identify the protein component of the BKV receptor and to develop pharmaceutical agents capable of mitigating BKV entry into human renal proximal tubular epithelial cells (HRPTEC), considered to be one of the main natural targets of BKV. The identity of the BKV receptor is unknown. At present, no specific pharmacological agent preventing BKV nephritis is available. Currently, the only efficient therapy against BKV nephritis appears to be a reduction/change of immunosuppressive agents, and this may increase the inherent risk of rejection. In our preliminary studies we have established that caveolar endocytosis is critical for BKV infection of HRPTEC and revealed several important steps of the BKV intracellular trafficking pathway in HRPTEC. Furthermore, our data suggest that the antagonist of Endothelin-1 binding to Endothelin B receptor (ETRB) prevents BKV infection of HRPTEC. Endothelins (ET), agonists of G- protein coupled receptors; act as important mediators of renal disease progression. Our working hypothesis is that binding of BKV to ETRB in HRPTEC is the critical step in the BKV entry into HRPTEC and that ETRB antagonists can be efficient as agents against BKV nephritis. Specific Aim 1 will test the hypothesis that N- linked glycoprotein ETBR serves as a receptor for BKV particles in HRPTEC. To establish that ETRB serves as the binding molecule for BKV particles in HRPTEC, siRNA mediated silencing of the ETRB gene will be employed. We will also carry out experiments to determine direct association between ETRB and BKV particles and evaluate necessity for ETRB presence for BKV infection. BKV infection will be detected using the following markers: the percentage of infected cells, as detected by immunofluorescence, the cellular levels of BKV large T antigen expression, as detected by western blot analysis and viral load, as detected by real-time PCR. Proposed study will provide the basis for a novel strategy for therapeutical intervention in BKN, ultimately improving long-term graft survival after renal transplantation. PUBLIC HEALTH RELEVANCE: In recent years nephritis induced by BK virus (BKV), non-enveloped double-strand deoxyribonucleic acid polyomavirus, has become a severe problem after renal transplantation. Polyomavirus nephropathy, also termed BK-virus nephropathy (BKN) affects 1% to 10% of all kidney transplant recipients and is emerging as an escalating threat. Since graft loss due to BKN ranged from 10% to more than 80% in kidney transplant recipients with BKN there is a critical need now to better define the molecular mechanisms of BKV entry into its target cells and to develop efficient treatment strategies of BKN. The identity of the BKV receptor is unknown and no specific pharmacological agent preventing BKV nephritis is available. This proposal will uncover the protein component of BKV receptor and provide the basis for a novel strategy for therapeutical intervention in BKN to ultimately improve long-term graft survival after renal transplantation.