Neuroimmune regulation of neurotropic JC virus by SF2/ASF in glial cells

描述（由申请方提供）：进行性多灶性白质脑病（PML）是一种由人类嗜神经JC病毒引起的致死性脱髓鞘疾病。JCV在儿童时期感染超过80%的人群，并在余生中建立潜伏感染。 该病毒在免疫调节条件下以未知机制重新激活，导致中枢神经系统（CNS）中少突胶质细胞和星形胶质细胞的生产性感染。JCV几乎仅在神经胶质细胞中复制，并且其具有组织特异性特征的启动子序列通过与细胞因子的通讯在适当的细胞类型和免疫条件下紧密调节病毒基因组的表达。我们最近确定了选择性剪接因子SF2/ASF，作为JCV的潜在调节因子，因为其在神经胶质细胞中的过表达强烈抑制病毒基因表达和复制。 出乎意料的是，SF 2/ASF下调JCV是在转录阶段介导的，因此归因于SF 2/ASF在控制启动子活性中的新作用。 我们的初步研究结果表明，PBMC分泌的免疫介质诱导SF 2/ASF的表达，并抑制JCV的复制。 这些观察结果 提示在外周免疫细胞和神经胶质细胞之间存在一种新的免疫信号传导途径，该途径在病毒再活化过程中控制JCV基因表达的立即早期阶段。我们对JCV感染的神经胶质细胞的初步研究也揭示了一种重要的作用，即一种对JCV复制至关重要的小病毒蛋白，在SF2/ASF的调控中。SF 2/ASF的表达被诱导，并与感染细胞的细胞质中的未知蛋白共定位。生化分析结果表明，与SF 2/ASF的相互作用，这两种蛋白质之间的功能交叉关联的未知蛋白。报告基因分析结果表明，未知蛋白诱导SF2/ASF沉默的病毒转录。这些观察结果为我们研究SF2/ASF在JCV生命周期中的作用提供了理论基础，并使我们形成了我们的中心假设，即JCV复制的神经免疫调节是由SF2/ASF介导的，而病毒未知蛋白干扰这种调节。我们建议检查我们的假设，（i）检查免疫介导的调节SF2/ASF和复制的JCV在潜伏期以及生产期的病毒感染和（ii）调查SF2/ASF和JCV未知蛋白之间的功能相互作用，在这种调节。我们将采用分子、细胞和病毒学方法来解决这些问题，并通过对PML患者临床样本的免疫组化评价来研究我们发现的生物学相关性。这些研究的信息将为PML的治疗提供新的策略。

DESCRIPTION (provided by applicant): Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by human neurotropic JC virus. JCV infects more than 80% of human population during the childhood and establishes a latent infection for the rest of the life. The virus reactivates under immunomodulatory conditions with an unknown mechanism, resulting in productive infection of oligodendrocytes and astrocytes in the central nervous system (CNS). JCV replicates almost exclusively in glial cells, and its promoter sequence, which has tissue- specific characteristics, tightly modulates expression of viral genome in appropriate cell types and immune conditions through communication with cellular factors. We recently identified the alternative splicing factor, SF2/ASF, as a potential regulator of JCV as its overexpression in glial cells strongly suppresses viral gene expression and replication. Unexpectedly, down-regulation of JCV by SF2/ASF is mediated at transcriptional stage, thus ascribing a novel role for SF2/ASF in the control of promoter activity. Results from our preliminary studies suggest that immune mediators secreted by PBMCs induce the expression of SF2/ASF, and inhibit the replication of JCV. These observations suggest operation of a novel immune signaling pathway between peripheral immune cells and glial cells that controls the immediate early stage of JCV gene expression during the course of viral reactivation. Our preliminary studies from JCV-infected glial cells also revealed a significant role of agnoprotein, a small viral protein crucial for JCV replication, in regulation of SF2/ASF. Expression of SF2/ASF is induced, and colocalized with agnoprotein in the cytoplasm of infected cells. Results from biochemical assays showed interaction of agnoprotein with SF2/ASF suggesting a functional cross-association between these two proteins. Results from reporter gene assays revealed that agnoprotein induces the viral transcription silenced by SF2/ASF. These observations provided us a rationale to study the role of SF2/ASF in JCV life cycle, and led us to formulate our central hypothesis which s that neuroimmune regulation of JCV replication is mediated by SF2/ASF, and viral agnoprotein interferes with this regulation. We propose to examine our hypothesis by (i) examining immune mediated regulation of SF2/ASF and replication of JCV during the latent as well as productive period of the viral infection and (ii) investigating the functional interplay between SF2/ASF and JCV agnoprotein in this regulation. We will employ molecular, cellular, and virological approaches to address these questions and examine the biological relevance of our findings by immunohistochemical evaluation of clinical samples from patients with PML disease. The information from these studies will offer a novel strategy for the treatment of PML.