Regulatory Roles of Agnoprotein in Biology of JC virus

 描述(申请人提供)：JC病毒(JCV)导致中枢神经系统(CNS)的一种致命疾病，称为进行性多灶性白质脑病(PML)，患者患有潜在的免疫抑制状况，包括白血病、淋巴瘤和艾滋病。PML在艾滋病流行时代急剧增加，是一种与艾滋病相关的疾病。这种疾病在自身免疫性疾病患者中也在稳步增加，例如多发性硬化症和克罗恩病，他们使用基于抗体的药物(Natalizumab)进行治疗，这使得JCV成为自身免疫性疾病人群的风险因素。我们的实验室在研究JCV独特的生物学方面有多年的丰富经验。然而，近年来，我们将注意力集中在其调控蛋白之一的Agnotein(Agno)(71aaLong)的功能研究上，该蛋白被证明在病毒生命周期中发挥关键作用。在缺乏表达的情况下，JCV无法维持其生产周期。近年来，我们在理解AgNO的生物学功能方面取得了重大进展。例如，(I)在体外和体内，AgNO被发现形成高度稳定的二聚/低聚(多聚体)结构，这是由跨度从28到39的氨基酸介导的，这形成了我们最近的核磁共振研究证实的两亲性α-螺旋。(2)AgNO经历快速降解过程，如果通过诱变改变α-螺旋区域，病毒复制水平显著降低。(Iii)有趣的是，二聚结构域突变体在病毒转录本的选择性剪接过程中存在严重缺陷，这表明AgNO在病毒RNA剪接调控中发挥了新的作用。最初对二聚化结构域突变体的剪接研究甚至导致了JCV晚期转录本的一个新的和先前未预测的开放阅读框架(新的ORF)的发现，其重要性需要在JCV复制周期中被揭示。(4)此外，AgNO还被发现与JCV RNA特异性结合，并含有预测的核输出信号(NES)。此外，如果用CRM1特异性抑制剂Leptomycin B处理，它与CRM1特异地相互作用并在感染细胞的细胞核中积聚。这些发现还表明，AgNO在CRM1依赖的病毒RNA核质输出中发挥了新的调节作用。综上所述，这些发现为我们进一步研究AgNO在JCV转录产物的剪接和核质输出中的新调控作用提供了强有力的理论基础；并使我们能够形成我们的中心假设：AgNO及其多聚体在病毒转录产物的选择性剪接及其依赖于CRM1的核质输出中发挥着重要的调节作用。我们建议通过深入研究AgNO调控这两个事件的分子机制(S)来检验我们的假设。1

 DESCRIPTION (provided by applicant): JC virus (JCV) causes a fatal disease in the central nervous system (CNS), known as progressive multifocal leukoencephalopathy (PML) in patients with underlying immunosuppressive conditions, including leukemia, lymphoma and AIDS. PML in the era of the AIDS epidemic dramatically increased and is an AIDS associated illness. This disease is also steadily increasing among patients with autoimmune disorders, such as multiple sclerosis and Crohn's disease, who are treated with antibody-based drugs (natalizumab), which makes JCV as a risk factor for autoimmune disease populations. Our lab has considerable experience in studying the unique biology of JCV for a number of years. In recent years, however, we have focused our attention to the investigation of the functional roles of one of its regulatory proteins, Agnoprotein (Agno) (71 aa long), which is shown to play critical roles in the viral life cycle. In the absence of its expression, JCV is unable to sustain its productive cycle. We have made significant advances in understanding the biological functions of Agno in recent years. For instance, (i) Agno was found to form highly stable dimeric/oligomeric (multimeric) structures in vitro and in vivo and this is mediated by amino acids spanning from 28 to 39 which forms an amphipathic alpha-helix confirmed by our recent NMR studies. (ii) Agno undergoes a rapid degradation process and the viral replication levels significantly decrease if the alpha- helix region is altered by mutagenesis. (iii) Interestingly, the dimerization domain mutants were found to have profound defects in the alternative splicing process of the viral transcripts, suggesting new roles for Agno in regulation of viral RNA splicing. The initial splicing studies wit the dimerization domain mutants even led to the discovery of a novel and previously unpredicted open reading frame (new ORF) for JCV late transcripts, the significance of which needs to be uncovered for the JCV replication cycle. (iv) Moreover, Agno was also found to specifically bind to the JCV RNA and harbors a predicted nuclear export signal (NES). Furthermore, it specifically interacts with CRM1 and accumulates in the nucleus of the infected cells if they are treated with a CRM1 specific inhibitor, Leptomycin B. These findings also suggest novel regulatory roles for Agno in nucleo- cytoplasmic export of the viral RNA in a CRM1-dependent manner. Collectively, these findings provide us with a strong rationale to further investigate the novel regulatory roles of Agno in splicing and the nucleo- cytoplasmic export of JCV transcripts; and allowed us formulate our central hypothesis: Agno and its multimeric forms play important regulatory roles in the alternative splicing of the viral transcrips and their CRM1-dependent nucleo-cytoplasmic export. We propose to examine our hypothesis by thoroughly investigating the molecular mechanism(s) of the regulation of the both events by Agno. 1