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

描述(申请人提供)：进行性多灶性白质脑病(PML)是一种由人类嗜神经性JC病毒引起的致死性脱髓鞘疾病。JCV在儿童时期感染了80%以上的人口，并建立了一种终身潜伏感染。该病毒在免疫调节条件下重新激活，机制未知，导致中枢神经系统(CNS)中的少突胶质细胞和星形胶质细胞的生产性感染。JCV几乎只在胶质细胞中复制，其启动子序列具有组织特异性，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控病毒基因组的表达。我们最近发现选择性剪接因子SF2/ASF是JCV的潜在调节因子，因为它在神经胶质细胞中的过度表达强烈抑制病毒基因的表达和复制。出人意料的是，SF2/ASF对JCV的下调是在转录阶段介导的，因此SF2/ASF在控制启动子活性方面发挥了新的作用。 初步研究结果表明，PBMC分泌的免疫介质可诱导SF2/ASF的表达，并抑制JCV的复制。这些观察结果 建议在外周免疫细胞和神经胶质细胞之间运行一条新的免疫信号通路，在病毒重新激活过程中控制JCV基因表达的即刻早期阶段。我们对JCV感染的神经胶质细胞的初步研究也揭示了无核蛋白在调节SF2/ASF中的重要作用，这是一种对JCV复制至关重要的小病毒蛋白。SF2/ASF被诱导表达，并与不可知蛋白共存于感染细胞的细胞质中。生化分析结果表明，无核蛋白与SF2/ASF相互作用，表明这两种蛋白之间存在功能上的交叉结合。报告基因分析结果表明，未知蛋白诱导病毒转录被SF2/ASF沉默。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，并导致我们提出了S的中心假说，即JCV复制的神经免疫调节是由Sf2/Asf介导的，病毒未知蛋白干扰了这一调节。我们建议通过(I)检测病毒感染潜伏期和产生期对SF2/ASF和JCV复制的免疫调节以及(Ii)研究SF2/ASF和JCV无核蛋白在这一调节中的功能相互作用来验证我们的假设。我们将使用分子、细胞和病毒学方法来解决这些问题，并通过对PML患者的临床样本进行免疫组织化学评估来检验我们的发现的生物学相关性。这些研究的信息将为PML的治疗提供一种新的策略。

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.