Ramphan Suwipa, Khongwichit Sarawut, Saisawang Chonticha, Kovanich Duangnapa, Ketterman Albert J, Ubol Sukathida, Auewarakul Prasert, Roytrakul Sittiruk, Smith Duncan R, Kuadkitkan Atichat
Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Proteomics Clin Appl. 2018 Jul;12(4):e1700020. doi: 10.1002/prca.201700020. Epub 2017 Nov 16.
Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes.
To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis.
A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels.
These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.
基孔肯雅病毒（CHIKV）是一种通过蚊子传播的甲病毒，可引起人类基孔肯雅热。CHIKV非结构蛋白2（nsP2）是一种多功能蛋白，它还能调节宿主细胞以抑制先天性免疫反应并抑制其他细胞过程。
为了进一步研究nsP2与宿主细胞的相互作用，将CHIKV nsP2的蛋白酶结构域（nsP2-pro）转染到Hela细胞中，并通过二维聚丙烯酰胺凝胶电泳检测差异蛋白表达。
共观察到21个差异调节的斑点（6个上调，15个下调），其中5个通过质谱鉴定。通过对nsP2-pro转染和CHIKV自然感染进行蛋白质印迹，证实了其中一种已鉴定蛋白泛素结合酶E2 L3（UBE2L3）的下调，并且还表明UBE2L3的下调需要具有酶活性的nsP2蛋白酶结构域。在CHIKV感染之前将全长UBE2L3转染到HEK293T/17细胞中可降低感染水平和E蛋白表达，但不会改变RNA基因组水平。
这些结果表明UBE2L3是CHIKV nsP2蛋白酶的细胞靶点，这可能介导了基孔肯雅热的发病机制。
