Distribution and function of JCV agnoprotein
Distribution and function of JCV agnoprotein
复制标题
JCV不可知蛋白的分布和功能
DOI：
10.1080/13550280152537148
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发表时间：
2001
影响因子：
3.2
通讯作者：
K. Nagashima
中科院分区：
文献类型：
--
作者：
Y. Okada;S. Endo;Hidehiro Takahashi;H. Sawa;T. Umemura;K. Nagashima
JC病毒（JC virus，JCV）是导致进行性多灶性白质脑病（progressive multifocal leukoencephalopathy，PML）的病原体，编码包括未知蛋白在内的6种主要蛋白质，其功能尚不清楚。为了探讨其功能，我们用免疫组织化学方法初步研究了该蛋白在培养细胞和PML脑组织中的表达和定位。采用特异性多克隆抗体，主要是在持续感染的JCI细胞的细胞质中，并在PML脑中的少突胶质细胞的精细加工的细胞质过程中发现了未知蛋白。免疫组化显示脱髓鞘灶内少突胶质细胞胞浆保存较好。Agnoprotein与微管蛋白共沉淀的免疫沉淀试验和共定位的Agnoprotein与细胞质微管蛋白的双重免疫染色与共聚焦显微镜进行了验证。将未知基因缺失的JCV Mad 1株（Mad 1 Δ阿尼奥）转染到易感细胞系中不仅不能产生未知蛋白，而且不能产生VP 1和大T mRNA，而野生型JCV Mad 1导致大T和VP 1 mRNA的表达。胞质内的未知蛋白被磷酸化，当与GST-EGFP共表达时，也定位于胞质中。蛋白激酶A的抑制剂H-89的抑制，但是，逆转了胞质定位的agnoprotein的核室。我们的研究结果表明，在病毒复制过程中，JCV未知蛋白可能以磷酸化依赖的方式在细胞核和细胞质之间“穿梭”。
JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), encodes six major proteins including agnoprotein, the function of which is unknown. To explore its function, we initially studied the expression and localization of agnoprotein in both cultured cells and PML brain using immunohistochemical methods. Employing a specific polyclonal antibody, agnoprotein was found mostly in the cytoplasm of persistently infected JCI cells and in the finely elaborated cytoplasmic processes of oligodendroglial cells in PML brain. The immunohistochemistry indicated that the cytoplasm of oligodendroglial cells was relatively well-preserved in the demyelinated foci. Agnoprotein coprecipitated with tubulin in immunoprecipitation assays and the colocalization of agnoprotein with cytoplasmic tubulin was verified by double immunostaining with confocal microscopy. Transfection of an agnogene deleted JCV Mad1 strain (Mad1Δagno) into the susceptible cell line failed to produce not only agnoprotein but also VP1 and large T mRNAs, whereas the wild-type JCV Mad1 resulted in the expression of both large T and VP1 mRNAs. The cytoplasmic agnoprotein was phosphorylated and when coexpressed with GST-EGFP, was also localized in the cytoplasm. Inhibition of protein kinase A by its inhibitor H-89, however, reversed the cytoplasmic localization of agnoprotein to the nuclear compartment. Our results suggestthat JCV agnoprotein may “shuttle” between the nucleus and cytoplasm in a phosphorylation-dependent manner during viral replication.