Migration of polyomavirus JC across the blood-brain-barrier

描述（由申请方提供）：由人多瘤病毒JC（JCV）引起的进行性多灶性白质脑病（PML）仍然是HIV/AIDS患者发病和死亡的重要原因，即使在HAART后时代。JCV进入中枢神经系统（CNS）的潜在机制尚不清楚。也就是说，目前尚不清楚无细胞JCV是否使用唾液酸受体（SAR）感染人脑微血管内皮（HBMVE）细胞，或者JCV感染的B细胞是否改变细胞粘附分子（CAM）的表达模式，如极晚期抗原-4（VLA-4）和淋巴细胞功能相关抗原-1（LFA-1），以及趋化因子受体CCR 2和CXCR 1/CXCR 2，以优先穿过BB B。我们的初步和发表的数据表明，JCV生产性地感染原代HBMVE细胞;无细胞JCV穿过体外BBB模型;感染性JCV病毒粒子可以潜伏地存活在Epstein巴尔病毒转化的B细胞中;并且来自B细胞的潜伏JCV可以感染原代人胎儿神经胶质（PHFG）细胞。然而，关于JCV感染的B细胞穿过BB B的迁移的数据是不可用的。基于这些数据，我们将检验以下假设：1）无细胞JCV利用SAR感染HBMVE细胞，并穿过体外BB B; 2）潜伏感染的B细胞通过调节CAM和趋化因子受体的表达而优先穿过BB B。所提出的研究将通过解决两个具体目标来具体解决我们对无细胞和B细胞相关JCV跨BB B的迁移机制的认识中的差距：具体目标1：通过记录HBMVE细胞中JCV感染和复制的变化，表征无细胞JCV与HBMVE细胞和体外BBB的相互作用，和当用SAR阻断剂治疗时JCV穿过BBB的迁移，以及在JCV迁移之前、期间和之后通过监测连接蛋白、claudin-1和claudin-5和occludin、TEER和细胞旁标志物（如菊粉或葡聚糖）的表达来监测BBB的变化。具体目标二：通过用JCV感染B细胞并比较JCV感染和未感染的B细胞上粘附分子VLA-4和LFA-1以及趋化因子受体CCR 2和CXCR 1/CXCR 2的表达，以及比较JCV感染和未感染的B细胞穿过体外BBB的迁移，来证明和表征JCV感染的B细胞穿过体外BBB的运输，并记录在迁移之前、期间和之后体外BBB的变化。中枢神经系统感染很难治疗，因为大脑周围有各种障碍。拟议的研究应该提供新的见解，紧密连接蛋白和CAM如何变得失调，并进一步促进JCV穿过BBB的迁移，以及特异性抑制剂如何阻止JCV进入和/或感染HBMVE细胞，星形胶质细胞和少突胶质细胞。新发现的知识可能会改善PML的治疗干预。由人类多瘤病毒JC（JCV）引起的进行性多灶性白质脑病（PML）是一种致命的脑部疾病，仍然是免疫系统缺陷人群死亡和发病的重要原因之一。目前，尚无可用于管理PML患者的预防或治疗选择。拟议的研究将利用体外系统来描绘JCV穿越血脑屏障的细胞和分子步骤，这些机制的发现可能有助于我们为这种不治之症制定预防或治疗干预措施。

DESCRIPTION (provided by applicant): Progressive multifocal leukoencephalopathy (PML), caused by human polyomavirus JC (JCV), remains an important cause of morbidity and mortality among HIV/AIDS patients even in the post-HAART era. The underlying mechanism(s) of JCV entry into the central nervous system (CNS) is poorly understood. That is, it is unclear if cell-free JCV uses sialic acid receptor (SAR) to infect human brain microvascular endothelial (HBMVE) cells or if JCV-infected B cells alter the expression patterns of cell adhesion molecules (CAM), such as very late antigen-4 (VLA-4) and lymphocyte function associated-antigen-1 (LFA-1), and chemokine receptors, CCR2 and CXCR1/CXCR2, to preferentially cross the BBB. Our preliminary and published data demonstrate that JCV productively infects primary HBMVE cells; cell-free JCV crosses the in-vitro BBB model; infectious JCV virions can survive latently in Epstein Barr virus-transformed B cells; and latent JCV from B cells can infect primary human fetal glial (PHFG) cells. However, data on the transmigration of JCV-infected B cells across the BBB are unavailable. Based on these data, we will test the following hypotheses: 1) cell-free JCV employs the SAR to infect HBMVE cells, and to cross the in-vitro BBB; 2) latently infected B cells preferentially cross the BBB by modulating of the expression of CAM and chemokine receptors. The proposed research will specifically address gaps in our knowledge about the mechanism of transmigration of cell-free and B cell- associated JCV across the BBB by addressing two specific aims: Specific Aim 1: To characterize the interaction of cell-free JCV with the HBMVE cells and the in-vitro BBB, by documenting changes in infection and replication of JCV in HBMVE cells, and JCV transmigration across the BBB when treated with SAR blockers, and changes in BBB by monitoring the expression of junctional proteins, claudin-1 and -5 and occludin, TEER, and paracellular markers, such as inulin or dextran, before, during and after transmigration of JCV. Specific Aim 2: To demonstrate and characterize the trafficking of JCV-infected B cells across the in-vitro BBB by infecting B cells with JCV and comparing the expression of adhesion molecules VLA-4 and LFA-1 and chemokine receptors CCR2 and CXCR1/CXCR2 on JCV-infected and -uninfected B cells, and comparing the transmigration of JCV-infected and -uninfected B cells across the in-vitro BBB, and documenting changes in the in-vitro BBB before, during and after transmigration. CNS infections are difficult to treat because of the various barriers surrounding the brain. The proposed study should provide novel insights into how tight junction proteins and CAM become dysregulated and further facilitate migration of JCV across the BBB, and how specific inhibitors can block JCV from entering and/or infecting HBMVE cells, astrocytes and oligodendrocytes. Newfound knowledge may lead to improved therapeutic interventions for PML. PUBLIC HEALTH RELEVANCE Progressive multifocal leukoencephalopathy (PML), a fatal brain disease, caused by human polyomavirus JC (JCV) remains one of the important causes of mortality and morbidity among people with defective immune systems. Currently, there are no preventive or therapeutic options available to manage PML patients. The proposed research will utilize in-vitro systems to delineate cellular and molecular steps involved in JCV transmigration across the blood-brain-barrier and the discovery of such mechanisms may assist us in developing preventive or therapeutic intervention for this incurable disease.