BHK-21 세포에서의 일본뇌염바이러스 구조단백질에 의한 세포독성

Cytopathic Effects of Japanese Encephalitis Virus Structural Proteins in BHK-21 Cells

일본뇌염바이러스(Japanese encephalitis virus, JEV)의 구조단백질 capsid (C), precursor membrane (prM/M), 및 envelop (E) 단백질의 독립적인 발현을 위한 inducible expression system을 구축하였다. 발현세포주로는 BHK-21을 사용하였으며 발현의 induction에는 tetracycline analog인 doxycycline이 사용되었다. Transfectant BHK-21/IV(vector대조구), BHK21/IC(C), BHK-21/IP (prM/M),및 BHK-21/IE는 G418과 hygromycin 존재하에 클로닝되었으며 doxycycline induction에 따른 각 유전자의 mRNA 전사를 확인하였다. 세포의 성장곡선, chromatin condensation, internucleosomal DNA fragmentation, 및 flow cytometry에 의한 DNA content profile 분석을 통해 induction에 의한 각 구조단백질의 발현이 숙주세포에 미치는 영향을 조사하였다. 세 transfectants 모두 세포성장이 감소하고 chromatin이 응축되었다. 그러나 DNA fragmentation 및 DNA content profile 분석에서는BHK-21/IC만이 induction에 따라 상응하여 반응하였다. 이상의 결과는 JEV 감염에 의한 apoptotic 세포사멸 유도기전에서 capsid 단백질이 직접적이고 독립적인 영향요인이 될 수 있음을 제시한다.

Inducible expression system for the three structural proteins, capsid (C), precursor membrane (prM/M), and envelop (E) of Japanese encephalitis virus (JEV) was established in BHK-21 cells. Doxycycline, a tetracycline analog, was utilized as an inducer. Transfectants BHK-21/IV (vector only), BHK-21/IC (for C), BHK-21/IP3 (for prM), and BHK-21/IE1 (for E) were selected and cloned in the presence of G4l8 or hygromycin. Transcribed mRNAs for the corresponding genes were observed after doxycycline induction. Effects by the JEV structural gene expression on the transfectants were monitored via cell growth, chromatin condensation, internucleosomal DNA fragmentation, and DNA contents analyses. Clear cell growth retardation and chromatin condensation were observed in all three transfectants while only BHK-2/IC corresponded to the induction status in the DNA fragmentation and DNA content analyses. Combined results, therefore, suggested that JEV capsid protein should be one of the direct and independent factors in apoptotic cell death induced by IEV infection.