BK Virus Receptor and polyomavirus nephropathy

描述（由申请人提供）：近年来，BK病毒（BKV）（一种无包膜双链DNA多瘤病毒）诱导的肾炎已成为肾移植后的严重问题。迫切需要更好地定义BKV进入其靶细胞的分子机制，并开发BKN的有效治疗策略。拟议研究的目的是鉴定BKV受体的蛋白组分，并开发能够减轻BKV进入人肾近端肾小管上皮细胞（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肾炎的特异性药物。目前，对BKV肾炎的唯一有效治疗似乎是减少/改变免疫抑制剂，这可能会增加排斥反应的固有风险。在我们的初步研究中，我们已经确定，小窝内吞作用是至关重要的BKV感染的HRPTEC，并揭示了几个重要的步骤的BKV细胞内运输途径在HRPTEC。此外，我们的数据表明，内皮素-1结合内皮素B受体（ETR B）的拮抗剂防止BKV感染HRPTEC。内皮素（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BKV与HRPTEC中ETRB的结合是BKV进入HRPTEC的关键步骤，并且ETRB拮抗剂可以有效地作为抗BKV肾炎的药剂。具体目标1将检验N-连接糖蛋白ETBR作为HRPTEC中BKV颗粒的受体的假设。为了确定ETRB作为HRPTEC中BKV颗粒的结合分子，将采用siRNA介导的ETRB基因沉默。我们还将进行实验，以确定ETRB和BKV颗粒之间的直接关联，并评估ETRB存在对BKV感染的必要性。将使用以下标志物检测BKV感染：感染细胞的百分比（通过免疫荧光检测）、BKV大T抗原表达的细胞水平（通过蛋白质印迹分析检测）和病毒载量（通过实时PCR检测）。拟议的研究将为BKN的治疗干预提供新策略的基础，最终提高肾移植后移植物的长期存活率。 公共卫生相关性：BK病毒（BKV）是一种无包膜的双链脱氧核糖核酸多瘤病毒，近年来引起的肾炎已成为肾移植术后的严重问题。多瘤病毒肾病，也称为BK病毒肾病（BKN），影响所有肾移植受者的1%至10%，并正在成为一个不断升级的威胁。由于在患有BKN的肾移植受者中，由于BKN引起的移植物损失范围从10%到超过80%，因此现在迫切需要更好地定义BKV进入其靶细胞的分子机制，并开发BKN的有效治疗策略。BKV受体的特性尚不清楚，也没有预防BKV肾炎的特异性药物。该提案将揭示BKV受体的蛋白组分，并为BKN的治疗干预提供新策略的基础，以最终改善肾移植后的长期移植物存活率。

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.