Regulatory Roles of Agnoprotein in Biology of JC virus

Agno蛋白在JC病毒生物学中的调节作用

• 批准号: 8922270
• 负责人: MAHMUT SAFAK
• 金额: $ 39万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8922270
• 关键词: Acquired Immunodeficiency Syndrome; Alternative Splicing; Amino Acids; Amphipathic Alpha Helix; Antibodies; Applications Grants; Attention; Autoimmune Diseases; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Biological Process; Biology; Brain; Cell Nucleus; Cells; Central Nervous System Diseases; Clinical; Code; Crohn' s disease; Cytoplasm; DNA biosynthesis; Defect; Dimerization; Disease; Elements; Epidemic; Event; Genetic; Goals; Grant; HIV-1; Immunocompromised Host; Immunosuppressive Agents; In Vitro; Incidence; Individual; Investigation; JC Virus; Large T Antigen; Leucine; Life Cycle Stages; Maintenance; Maps; Mediating; Messenger RNA; Molecular; Multiple Sclerosis; Mutagenesis; Mutation; Myelin; Neuraxis; Neuroglia; Neurologic Manifestations; Nuclear Export; Oligodendroglia; Open Reading Frames; Patients; Pattern; Pharmaceutical Preparations; Play; Population; Process; Progressive Multifocal Leukoencephalopathy; Protein Export Pathway; RNA; RNA Recognition Motif; RNA Sequences; RNA Splicing; Regulation; Reporting; Risk Factors; Role; Signal Transduction; Structure; Technology; Tertiary Protein Structure; Therapeutic; Transcript; Viral; Virus Replication; Wit; alpha helix; antigen binding; base; experience; genetic regulatory protein; in vivo; inhibitor/antagonist; insight; interest; latent infection; leptomycin B; leukemia/lymphoma; mutant; natalizumab; novel; patient population; protein function; protein protein interaction; public health relevance; rev Protein; transcriptome sequencing; viral DNA; viral RNA

 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

 描述（由申请方提供）：JC病毒（JCV）在中枢神经系统（CNS）中引起一种致死性疾病，在患有基础免疫抑制疾病（包括白血病、淋巴瘤和AIDS）的患者中称为进行性多灶性白质脑病（PML）。PML在艾滋病流行时代急剧增加，是一种艾滋病相关疾病。这种疾病在患有自身免疫性疾病的患者中也在稳步增加，如多发性硬化症和克罗恩病，他们用基于抗体的药物（那他珠单抗）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的独特生物学方面有相当多的经验。然而，近年来，我们已经把注意力集中在其调节蛋白之一，Agnoprotein（阿尼奥）（71 aa长），这是在病毒的生命周期中发挥关键作用的功能作用的调查。如果没有它的表达，JCV就无法维持其生产周期。近年来，我们在了解阿尼奥的生物学功能方面取得了重大进展。例如，（i）发现阿尼奥在体外和体内形成高度稳定的二聚体/寡聚体（多聚体）结构，这是由28至39个氨基酸介导的，这些氨基酸形成两亲性α-螺旋，这已由我们最近的核磁共振研究证实。(ii)如果通过突变改变α-螺旋区域，阿尼奥会经历快速降解过程，病毒复制水平会显着降低。(iii)有趣的是，发现二聚化结构域突变体在病毒转录物的选择性剪接过程中具有深刻的缺陷，这表明阿尼奥在调节病毒RNA剪接中的新作用。二聚化结构域突变体的初始剪接研究甚至导致发现了JCV晚期转录物的新的和先前未预测的开放阅读框架（新ORF），其意义需要被揭示用于JCV复制周期。(iv)此外，还发现阿尼奥与JCV RNA特异性结合，并具有预测的核输出信号（内斯）。此外，如果用CRM 1特异性抑制剂Leptomycin B处理受感染细胞，则它特异性地与CRM 1相互作用并在细胞核中积累。这些发现也表明阿尼奥在病毒RNA的核质输出中以CRM 1依赖性方式的新的调节作用。总的来说，这些发现为我们进一步研究阿尼奥在JCV转录物的剪接和核质输出中的新型调节作用提供了强有力的理论基础;并使我们能够制定我们的中心假设：阿尼奥及其多聚体形式在病毒转录物的选择性剪接及其CRM 1依赖性核质输出中发挥重要的调节作用。我们建议通过彻底研究阿尼奥对这两个事件的调节的分子机制来检验我们的假设。1