Agnoprotein in JC virus virion biogenesis and replication

JC病毒病毒体生物发生和复制中的Agno蛋白

• 批准号: 8113340
• 负责人: MAHMUT SAFAK
• 金额: $ 36.75万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8113340
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Antigens; Applications Grants; Astrocytes; Binding; Biogenesis; Brain; Capsid; Capsid Proteins; Cell Cycle Progression; Cells; Central Nervous System Diseases; Characteristics; Clinical; Code; Complex; DNA; DNA Binding; DNA biosynthesis; Disease; Disease Progression; Elements; Epidemic; Funding; Grant; Health; Human; Immunocompromised Host; Immunosuppressive Agents; In Vitro; Individual; Infection; Intake; JC Virus; Large T Antigen; Life Cycle Stages; Lymphoproliferative Disorders; Mediating; Mitosis; Molecular; Mutagenesis; Myelin; Myeloproliferative disease; Neuraxis; Oligodendroglia; PKC Phosphorylation Site; Patients; Phase Transition; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Polyomavirus; Process; Progressive Multifocal Leukoencephalopathy; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Regulation; Research Project Grants; Role; Testing; Therapeutic; Viral; Viral Regulatory Proteins; Virion; Virus Replication; central nervous system demyelinating disorder; deletion analysis; design; genetic regulatory protein; helicase; in vivo; latent infection; leukemia/lymphoma; mutant; neurotropic; research study; transcription factor; viral DNA

DESCRIPTION (provided by applicant): JC virus (JCV) is an etiological agent of Progressive Multifocal Leukoencephalopathy (PML), a fatal demyelinating disease of the Central Nervous System (CNS), which is frequently seen in patients with underlying immunosuppressive conditions, including leukemia, lymphomas and AIDS. PML in the era of AIDS epidemic dramatically increased which makes it an AIDS defining condition. JCV establishes a sub-clinical latent infection in the body but upon reactivation, it enters the brain; and lytically and abortively infects oligodendrocytes (myelin producing cells) and astrocytes respectively. This infection results in an extensive myelin loss in CNS, which is the characteristic histopathological landmark of PML. This is a revised competitive renewal R01 grant application, entitled "Role of agnoprotein in JC virus life cycle". During the course of this funding period, we established that agnoprotein of JCV plays important regulatory roles in JCV life cycle through molecular interactions with a cellular transcription factor, YB-1 and the viral regulatory protein, large T antigen. It deregulates cell cycle progression, in which agnoprotein positive cells largely accumulate at G2/M phase transition. We also demonstrated the involvement of the coding region of agnoprotein in regulation of JCV life cycle by deletion analysis. In this regard, we showed that agnoprotein- coding region contains important cis-acting DNA elements to which specific transcription factors bind and contribute the viral life cycle. Furthermore, analysis of the PKC phosphorylation sites of this protein by mutagenesis revealed the fact that phosphorylation plays a critical role in the function of agnoprotein, because none of the phosphorylation mutants (Ser7, Ser11 and Thr21 to Ala) was able to continue viral replication cycle due to a limited replication and empty capsid formation. Moreover, our recent findings also showed that phosphorylated form of agnoprotein is targeted by a Ser/Thr phosphatase, PP2A, for dephosphorylation and this can be inhibited by JCV small t antigen, suggesting a functional ternary complex formation between these three proteins. These findings collectively have provided us a strong rationale to further study the functions of agnoprotein and led us to hypothesize that agnoprotein plays critical regulatory roles in JCV virion biogenesis and therefore in the progression of PML. As such, the understanding of the molecular mechanisms in which agnoprotein is involved in JCV replication is central to unravel the molecular mechanisms that are critical for the disease progression so that we would be able to develop effective therapeutic strategies against this disease. Thus, to further examine our hypothesis, we propose to i) investigate the molecular mechanisms by which agnoprotein regulates both the virion formation and large T antigen-mediated viral DNA replication; and ii) examine the impact of both PP2A and Sm t-Ag on agnoprotein functions during this process. PUBLIC HEALTH RELEVANCE Progressive multifocal leukoencephalopathy (PML), a white mater disease of the central nervous system, is caused by a human neurotropic polyomavirus, JC virus (JCV). This disease mostly affects immunocompromised patients with underlying disorders such as lymphoproliferative and myeloproliferative diseases and acquired immunodeficiency syndrome, (AIDS). In this research project, we are proposing experiments to understand the role of one of JCV regulatory proteins, agnoprotein in viral biogenesis, which may allow us to design effective therapeutic strategies to curb the disease in affected individuals.

描述（由申请人提供）：JC 病毒（JCV）是进行性多灶性白质脑病（PML）的病原体，PML 是一种致命的中枢神经系统（CNS）脱髓鞘疾病，常见于患有潜在免疫抑制疾病的患者，包括白血病、淋巴瘤和艾滋病。在艾滋病流行时期，PML 急剧增加，使其成为艾滋病的定义条件。 JCV 在体内建立亚临床潜伏感染，但在重新激活后，它会进入大脑；并分别以溶解性和流产性方式感染少突胶质细胞（产生髓磷脂的细胞）和星形胶质细胞。这种感染导致中枢神经系统髓鞘质广泛丧失，这是 PML 的特征性组织病理学标志。这是修订后的竞争性更新 R01 拨款申请，题为“agno Protein 在 JC 病毒生命周期中的作用”。在此资助期间，我们确定 JCV 的无视蛋白通过与细胞转录因子 YB-1 和病毒调节蛋白大 T 抗原的分子相互作用，在 JCV 生命周期中发挥重要的调节作用。它解除了细胞周期进程的调节，其中不可知蛋白阳性细胞主要在 G2/M 相转变时积累。我们还通过缺失分析证明了agno蛋白的编码区参与JCV生命周期的调节。在这方面，我们表明agno蛋白编码区含有重要的顺式作用DNA元件，特定转录因子与这些元件结合并促进病毒生命周期。此外，通过诱变对该蛋白的 PKC 磷酸化位点进行分析揭示了磷酸化在无视蛋白的功能中起着关键作用的事实，因为由于复制有限和空衣壳形成，没有一个磷酸化突变体（Ser7、Ser11 和 Thr21 至 Ala）能够继续病毒复制周期。此外，我们最近的研究结果还表明，磷酸化形式的agno蛋白被Ser/Thr磷酸酶PP2A靶向去磷酸化，并且这可以被JCV小t抗原抑制，表明这三种蛋白之间形成功能性三元复合物。这些发现共同为我们进一步研究agno蛋白的功能提供了强有力的理由，并使我们假设agno蛋白在JCV病毒粒子的生物合成中发挥着关键的调节作用，因此在PML的进展中发挥着关键的调节作用。因此，了解agno蛋白参与JCV复制的分子机制对于阐明对疾病进展至关重要的分子机制至关重要，以便我们能够开发针对这种疾病的有效治疗策略。因此，为了进一步检验我们的假设，我们建议 i) 研究无视蛋白调节病毒体形成和大 T 抗原介导的病毒 DNA 复制的分子机制； ii) 在此过程中检查 PP2A 和 Sm t-Ag 对无视蛋白功能的影响。公共卫生相关性 进行性多灶性白质脑病 (PML) 是一种中枢神经系统白质疾病，由人类嗜神经性多瘤病毒 JC 病毒 (JCV) 引起。这种疾病主要影响患有淋巴增殖性疾病和骨髓增殖性疾病以及获得性免疫缺陷综合征（艾滋病）等基础疾病的免疫功能低下患者。在这个研究项目中，我们提出实验来了解 JCV 调节蛋白之一（无视蛋白）在病毒生物发生中的作用，这可能使我们能够设计有效的治疗策略来遏制受影响个体的疾病。