Kim Jin-Kyoung, Kim Jeong-Min, Song Byung-Hak, Yun Sang-Im, Yun Gil-Nam, Byun Sung-June, Lee Young-Min
Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
Department of Microbiology, College of Medicine, Chungbuk National University, Cheongju, South Korea.
PLoS One. 2015 Apr 27;10(4):e0124318. doi: 10.1371/journal.pone.0124318. eCollection 2015.
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is closely related to West Nile (WN), yellow fever (YF), and dengue (DEN) viruses. Its plus-strand genomic RNA carries a single open reading frame encoding a polyprotein that is cleaved into three structural (C, prM/M, and E) and at least seven nonstructural (NS1/NS1', NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins, based on previous work with WNV, YFV, and DENV. Here, we aimed to profile experimentally all the viral proteins found in JEV-infected cells. We generated a collection of 15 JEV-specific polyclonal antisera covering all parts of the viral protein-coding regions, by immunizing rabbits with 14 bacterially expressed glutathione-S-transferase fusion proteins (for all nine viral proteins except NS2B) or with a chemically synthesized oligopeptide (for NS2B). In total lysates of JEV-infected BHK-21 cells, immunoblotting with these antisera revealed: (i) three mature structural proteins (12-kDa C, ~8-kDa M, and ~53-kDa E), a precursor of M (24-kDa prM) and three other M-related proteins (10-14 kDa); (ii) the predicted ~45-kDa NS1 and its frameshift product, ~58-kDa NS1', with no evidence of the predicted ~25-kDa NS2A; (iii) the predicted but hardly detectable ~14-kDa NS2B and an unexpected but predominant ~12-kDa NS2B-related protein; (iv) the predicted ~69-kDa NS3 plus two major cleavage products (34-kDa NS3N-term and 35-kDa NS3C-term), together with at least nine minor proteins of ~16-52 kDa; (v) the predicted ~14-kDa NS4A; (vi) two NS4B-related proteins (27-kDa NS4B and 25-kDa NS4B'); and (vii) the predicted ~103-kDa NS5 plus at least three other NS5-related proteins (15 kDa, ~27 kDa, and ~90 kDa). Combining these data with confocal microscopic imaging of the proteins' intracellular localization, our study is the first to provide a solid foundation for the study of JEV gene expression, which is crucial for elucidating the regulatory mechanisms of JEV genome replication and pathobiology.
日本脑炎病毒（JEV）是一种由蚊子传播的黄病毒，与西尼罗河病毒（WN）、黄热病毒（YF）和登革病毒（DEN）密切相关。基于之前对西尼罗河病毒、黄热病毒和登革病毒的研究，其正链基因组RNA携带一个单一的开放阅读框，编码一个多聚蛋白，该多聚蛋白被切割成三种结构蛋白（C、prM/M和E）和至少七种非结构蛋白（NS1/NS1'、NS2A、NS2B、NS3、NS4A、NS4B和NS5）。在此，我们旨在通过实验分析JEV感染细胞中发现的所有病毒蛋白。我们通过用14种细菌表达的谷胱甘肽-S-转移酶融合蛋白（针对除NS2B外的所有九种病毒蛋白）或化学合成的寡肽（针对NS2B）免疫兔子，产生了一组覆盖病毒蛋白编码区所有部分的15种JEV特异性多克隆抗血清。在用这些抗血清对JEV感染的BHK-21细胞的总裂解物进行免疫印迹分析时发现：（i）三种成熟的结构蛋白（约12 kDa的C、约8 kDa的M和约53 kDa的E）、M的前体（约24 kDa的prM）和另外三种与M相关的蛋白（约10 - 14 kDa）；（ii）预测的约45 kDa的NS1及其移码产物约58 kDa的NS1'，未发现预测的约25 kDa的NS2A；（iii）预测的但几乎检测不到的约14 kDa的NS2B和一种意外的但占主导的约12 kDa的NS2B相关蛋白；（iv）预测的约69 kDa的NS3加上两种主要的切割产物（约34 kDa的NS3N端和约35 kDa的NS3C端），以及至少九种约16 - 52 kDa的次要蛋白；（v）预测的约14 kDa的NS4A；（vi）两种与NS4B相关的蛋白（约27 kDa的NS4B和约25 kDa的NS4B'）；（vii）预测的约103 kDa的NS5加上至少三种其他与NS5相关的蛋白（约15 kDa、约27 kDa和约90 kDa）。将这些数据与蛋白质细胞内定位的共聚焦显微镜成像相结合，我们的研究首次为JEV基因表达的研究提供了坚实的基础，这对于阐明JEV基因组复制和病理生物学的调控机制至关重要。
