Agnoprotein in JC virus virion biogenesis and replication

描述（由申请人提供）：JC病毒（JCV）是进行性多灶性脑白质病（PML）的病因，PML是一种致命的中枢神经系统脱髓鞘疾病，常见于潜在免疫抑制疾病的患者，包括白血病、淋巴瘤和艾滋病。在艾滋病流行的时代，PML急剧增加，使其成为艾滋病的决定性疾病。JCV在体内建立亚临床潜伏感染，但在重新激活后，它进入大脑；并分别裂解和流产感染少突胶质细胞（髓磷脂产生细胞）和星形胶质细胞。这种感染导致中枢神经系统广泛的髓磷脂丢失，这是PML的特征性组织病理学标志。这是一份修订的竞争性续期R01资助申请，题为“agnoprotein在JC病毒生命周期中的作用”。在此资助期间，我们确定了JCV的agno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agnoprotein阳性细胞主要积聚在G2/M阶段。我们还通过缺失分析证明了agnoprotein编码区参与了JCV生命周期的调控。在这方面，我们发现agno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并参与病毒的生命周期。此外，通过诱变分析该蛋白的PKC磷酸化位点揭示了磷酸化在agnoprotein的功能中起关键作用的事实，因为磷酸化突变体（Ser7， Ser11和Thr21 to Ala）由于复制有限和空衣壳的形成而无法继续病毒复制周期。此外，我们最近的研究结果还表明，磷酸化形式的agnoprotein被丝氨酸/苏氨酸磷酸酶PP2A靶向去磷酸化，并且可以被JCV小t抗原抑制，这表明这三种蛋白之间形成了功能性三元复合物。这些发现共同为我们进一步研究agnoprotein的功能提供了强有力的理论基础，并使我们假设agnoprotein在JCV病毒粒子的生物发生中起关键的调节作用，从而在PML的进展中起关键作用。因此，了解agnoprotein参与JCV复制的分子机制对于揭示对疾病进展至关重要的分子机制至关重要，这样我们就能够开发出有效的治疗策略来对抗这种疾病。因此，为了进一步检验我们的假设，我们建议：(1)研究agno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；ii)研究PP2A和Sm - t-Ag在这一过程中对agnoprotein功能的影响。进行性多灶性脑白质病（PML）是一种中枢神经系统白质疾病，由人类嗜神经多瘤病毒（JCV）引起。这种疾病主要影响免疫功能低下的患者，其基础疾病如淋巴细胞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，我们正在提出实验来了解JCV调节蛋白之一agnoprotein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抑制受影响个体的疾病。

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.