Zapata Heidi J, Nakatsugawa Masako, Moffat Jennifer F
Department of Microbiology and Immunology, State University of New York Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210, USA.
J Virol. 2007 Jan;81(2):977-90. doi: 10.1128/JVI.01470-06. Epub 2006 Nov 1.
The transcription factors ATF-2 and c-Jun are important for transactivation of varicella-zoster virus (VZV) genes. c-Jun is activated by the c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase pathway that responds to stress and cytokines. To study the effects of VZV on this pathway, confluent human foreskin fibroblasts were infected with cell-associated VZV for 1 to 4 days. Immunoblots showed that phosphorylated JNK and c-Jun levels increased in VZV-infected cells, and kinase assays determined that phospho-JNK was active. Phospho-JNK was detected after 24 h, and levels rose steadily over 4 days in parallel with accumulation of VZV antigen. The two main activators of JNK are MKK4 and MKK7, and levels of their active, phosphorylated forms also increased. The competitive inhibitor of JNK, SP600125, caused a dose-dependent reduction in VZV yield (50% effective concentration, congruent with 8 microM). Specificity was verified by immunoblotting; phospho-c-Jun was eliminated by 18 microM SP600125 in VZV-infected cells. Immunofluorescent confocal microscopy showed that phospho-c-Jun and most of phospho-JNK were in the nuclei of VZV-infected cells; some phospho-JNK was in the cytoplasm. MKK4, MKK7, JNK, and phospho-JNK were detected by immunoblotting in purified preparations of VZV virions, but c-Jun was absent. JNK was located in the virion tegument, as determined by biochemical fractionation and immunogold transmission electron microscopy. Overall, these results demonstrate the importance of the JNK pathway for VZV replication and advance the idea that JNK is a useful drug target against VZV.
转录因子ATF-2和c-Jun对水痘带状疱疹病毒（VZV）基因的反式激活很重要。c-Jun由c-Jun N端激酶（JNK）激活，JNK是丝裂原活化蛋白激酶途径的成员，对压力和细胞因子有反应。为了研究VZV对该途径的影响，将汇合的人包皮成纤维细胞用细胞相关的VZV感染1至4天。免疫印迹显示，VZV感染的细胞中磷酸化JNK和c-Jun水平升高，激酶测定确定磷酸化JNK具有活性。磷酸化JNK在24小时后被检测到，其水平在4天内与VZV抗原的积累平行稳步上升。JNK的两个主要激活剂是MKK4和MKK7，它们的活性磷酸化形式的水平也增加。JNK的竞争性抑制剂SP600125导致VZV产量呈剂量依赖性降低（50%有效浓度，相当于8 microM）。通过免疫印迹验证了特异性；18 microM SP600125可消除VZV感染细胞中的磷酸化c-Jun。免疫荧光共聚焦显微镜显示，磷酸化c-Jun和大部分磷酸化JNK位于VZV感染细胞的细胞核中；一些磷酸化JNK位于细胞质中。在纯化的VZV病毒粒子制剂中通过免疫印迹检测到MKK4、MKK7、JNK和磷酸化JNK，但未检测到c-Jun。通过生化分级分离和免疫金透射电子显微镜确定，JNK位于病毒粒子被膜中。总体而言，这些结果证明了JNK途径对VZV复制的重要性，并提出JNK是抗VZV的有用药物靶点这一观点。
