Sawa Hirofumi, Suzuki Tadaki, Orba Yasuko, Sunden Yuji, Nagashima Kazuo
Department of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Nishi 9, Kita 18, Kita-ku, Sapporo 060-0818, Japan.
No To Shinkei. 2007 Feb;59(2):101-8.
JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopathy and belongs to Polyomavirus. In this article we describe our recent research relating to this virus. First, JCV's major capsid protein VP1 possesses a nuclear localization signal (NLS) and has the ability to construct a virus-like particle (VLP). We have investigated the mechanism of nuclear entry of JCV using VLP, and clarified the role of NLS. In vitro transport assay revealed that wild type VLP (wtVLP), but not deltaNLSVLP, entered the nuclei of cells. The nuclear transport of wtVLP was dependent on the addition of importins alpha and beta and was prevented by antibodies to nuclear pore complex (NPC). These results suggested that JCV VLP binds to cellular importins via the NLS of VP1 and is transported into the nucleus through the NPC. Second, a yeast two-hybrid screen of a human brain cDNA library demonstrated that the fasciculation and elongation protein zeta 1 (FEZ1) and the heterochromatin protein lalpha (HPla) are proteins that interacted with JCV agnoprotein (Agno). In vitro binding assay showed that Agno interacts directly with FEZ1 and HPlalpha. We have also shown that Agno induces the dissociation of FEZ1 from microtubules and dissociates the interaction between HPlalpha and lamin B receptor. We have demonstrated that interaction between Agno and these host proteins inhibited nuclear egress of JCV. Third, in order to inhibit JCV infection in infected cells, we synthesized siRNA which is specific for JCV Agno. Immunoblotting and immunocytochemical analysis demonstrated that expression levels of agnoprotein and VP1 were significantly inhibited by specific siRNA. In addition, levels of viral mRNAs and viral production were decreased in the cells transfected with Agno siRNA. Furthermore, viral production of cell treated with Agno siRNA was significantly inhibited. These results indicate that post-infection treatment with siRNAs, that targets JCV Agno suppresses virus production in JCV infected cells. Thus, siRNA directed against JCV encoding genes may provide a useful tool for suppression of JCV infection.
JC病毒（JCV）是进行性多灶性白质脑病的病原体，属于多瘤病毒。在本文中，我们描述了我们最近关于这种病毒的研究。首先，JCV的主要衣壳蛋白VP1具有核定位信号（NLS），并具有构建病毒样颗粒（VLP）的能力。我们使用VLP研究了JCV进入细胞核的机制，并阐明了NLS的作用。体外转运试验表明，野生型VLP（wtVLP）而非缺失NLS的VLP进入细胞的细胞核。wtVLP的核转运依赖于导入蛋白α和β的添加，并被针对核孔复合体（NPC）的抗体所阻止。这些结果表明，JCV VLP通过VP1的NLS与细胞导入蛋白结合，并通过NPC转运到细胞核中。其次，对人脑cDNA文库进行的酵母双杂交筛选表明，成束和延伸蛋白ζ1（FEZ1）和异染色质蛋白1α（HP1α）是与JCV反式激活蛋白（Agno）相互作用的蛋白质。体外结合试验表明，Agno直接与FEZ1和HP1α相互作用。我们还表明，Agno诱导FEZ1从微管上解离，并使HP1α与核纤层蛋白B受体之间的相互作用解离。我们已经证明，Agno与这些宿主蛋白之间的相互作用抑制了JCV的核输出。第三，为了抑制受感染细胞中的JCV感染，我们合成了对JCV Agno特异的siRNA。免疫印迹和免疫细胞化学分析表明，特异性siRNA显著抑制了反式激活蛋白和VP1的表达水平。此外，在用Agno siRNA转染的细胞中，病毒mRNA水平和病毒产生量均降低。此外，用Agno siRNA处理的细胞的病毒产生受到显著抑制。这些结果表明，用靶向JCV Agno的siRNA进行感染后治疗可抑制JCV感染细胞中的病毒产生。因此，针对JCV编码基因的siRNA可能为抑制JCV感染提供一种有用的工具。
