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: 8302988
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8302988
• 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(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，常见于有潜在免疫抑制状况的患者，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，使其成为艾滋病的定义条件。JCV在体内建立了一种亚临床潜伏感染，但一旦重新激活，它就进入大脑；裂解和流产分别感染少突胶质细胞(髓鞘产生细胞)和星形胶质细胞。这种感染导致中枢神经系统广泛的髓鞘丢失，这是PML的组织病理学特征。这是一份经修订的竞争性续订R01拨款申请，题为“无核蛋白在JC病毒生命周期中的作用”。在这一资助过程中，我们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，证实了JCV的不可知蛋白在JCV的生命周期中发挥着重要的调节作用。它放松了对细胞周期进程的调控，在G2/M期转变时，不可知蛋白阳性细胞大量聚集。我们还通过缺失分析证明了无核蛋白编码区参与了JCV生命周期的调控。在这方面，我们发现无糖蛋白编码区包含重要的顺式作用DNA元件，特定的转录因子结合到这些元件上，并有助于病毒的生命周期。此外，通过突变对该蛋白的PKC磷酸化位点的分析表明，磷酸化在无核蛋白的功能中起着关键作用，因为磷酸化突变体(Ser7、Ser11和Thr21到Ala)由于复制有限和形成空衣壳而不能继续病毒复制周期。此外，我们最近的发现还表明，Ser/Thr磷酸酶PP2A可以靶向磷酸化形式的无核蛋白去磷酸化，而JCV Small t抗原可以抑制这种去磷酸化，这表明这三种蛋白之间形成了一个功能性的三元复合体。这些发现为我们进一步研究未知蛋白的功能提供了有力的理论基础，并使我们假设无核蛋白在JCV病毒粒子的生物发生中发挥着关键的调节作用，从而在PML的进展中发挥着关键的调节作用。因此，了解不可知蛋白参与JCV复制的分子机制是揭开疾病进展的关键分子机制的核心，以便我们能够开发有效的治疗策略来对抗这种疾病。因此，为了进一步验证我们的假设，我们建议：1)研究无核蛋白调节病毒粒子形成和大T抗原介导的病毒DNA复制的分子机制；2)研究PP2A和Smt-Ag在这一过程中对无核蛋白功能的影响。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。这种疾病主要影响免疫受损的患者，有潜在的疾病，如淋巴增生性和骨髓增生性疾病以及获得性免疫缺陷综合征(艾滋病)。在这个研究项目中，我们正在提议进行实验，以了解JCV调节蛋白之一，不可知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，以遏制受影响的个人的疾病。

项目成果

Human polyoma JC virus minor capsid proteins, VP2 and VP3, enhance large T antigen binding to the origin of viral DNA replication: evidence for their involvement in regulation of the viral DNA replication.

• DOI: 10.1016/j.virol.2013.10.031
• 发表时间: 2014-01-20
• 期刊: VIROLOGY
• 影响因子: 3.7
• 作者: Saribas, A. Sami;Mun, Sarah;Johnson, Jaslyn;El-Hajmoussa, Mohammad;White, Martyn K.;Safak, Mahmut
• 通讯作者: Safak, Mahmut

JC virus agnoprotein enhances large T antigen binding to the origin of viral DNA replication: evidence for its involvement in viral DNA replication.

• DOI: 10.1016/j.virol.2012.06.017
• 发表时间: 2012-11-10
• 期刊: Virology
• 影响因子: 3.7
• 作者: Saribas AS;White MK;Safak M
• 通讯作者: Safak M

Human polyomavirus JC small regulatory agnoprotein forms highly stable dimers and oligomers: implications for their roles in agnoprotein function.

• DOI: 10.1016/j.virol.2011.08.015
• 发表时间: 2011-11-10
• 期刊: Virology
• 影响因子: 3.7
• 作者: Saribas AS;Arachea BT;White MK;Viola RE;Safak M
• 通讯作者: Safak M