미생물학회지 (Korean Journal of Microbiology)

  • 제38권3호
  • /
  • Pages.213-220
  • /
  • 2002
  • /
  • 0440-2413(pISSN)
  • /
  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

BHK-21 세포에서의 일본뇌염바이러스 구조단백질에 의한 세포독성

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

  • 성기민 (경희대학교 생물학과 및 기초과학연구소) ;
  • 정용석 (경희대학교 생물학과 및 기초과학연구소)
  • 발행 : 2002.09.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

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.

키워드

참고문헌

  1. The Togaviridae and Flaviviridae. Replication of flaviviruses Brinton, M.A.;S. Schlesinger(ed.)
  2. J. Gen. Virol. v.70 Antigenic relationships between flaviviruses as determined by cross-neutralization tests Calisher, C.H.;N. Karabatsos;J.M. Dalrymple;R.E. Shope;J.S. Porterfield;E.G. Westway;W.E. Brandt https://doi.org/10.1099/0022-1317-70-1-37
  3. Annu. Rev. Microbiol. v.44 Flavivirus genome organization, expression and replication Chambers, T.J.;C.S. Hahn https://doi.org/10.1146/annurev.mi.44.100190.003245
  4. J. Virol. v.67 Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation and viral replication Chambers, T.J.;A. Nestorowicz
  5. EMBO J. v.16 Expression of the papillomavirus E2 protein in HeLa cells leads to apoptosis Desaintes, C.;C. Demeret;S. Goyat;M. Yaniv;F. Thierry https://doi.org/10.1093/emboj/16.3.504
  6. J. Gen. Virol. v.80 Measles virus-induced immunosuppression in vitro is associated with dregulation of G1 cell cycle control proteins Engelking, O.;L.M. Fedorov;R. Lilischkis;V. ter Meulen;S. Schneider-Schaulies https://doi.org/10.1099/0022-1317-80-7-1599
  7. J. Virol. v.65 Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of Dengue virus nonstructural proteins Falgout, B.;M. Pethel;Y.M. Zhang;C.J. Lai
  8. J. Gen. Virol. v.80 Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth Ghosh, A.K.;R. Steele;K. Meyer;R. Ray;R.B. Ray https://doi.org/10.1099/0022-1317-80-5-1179
  9. Science v.268 Transcriptional activation by tetracycline in mammalian cells Gossen, M.;S. Freundlieb;G. Bender;G. Muller;W. Hillen;H. Bujard https://doi.org/10.1126/science.7792603
  10. EMBO J. v.13 Functional interation between the HCMV IE2 transactivator and the retinoblastoma protein Hagemeier, C.;R. Caswell;G. Hayhurst;J. Sinclair;T. Kouzarides
  11. J. Biol. Chem. v.273 X-gene product of hepatitis virus induces apoptosis in liver cells Kim, H.T.;H.S. Lee;Y.D. Yun https://doi.org/10.1074/jbc.273.1.381
  12. J. Gen. Virol. v.77 Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein Kuo, M.D.;C. Chin;S.L. Hsu;J.Y. Shiao;T.M. Wang;J.H. Lin https://doi.org/10.1099/0022-1317-77-9-2077
  13. Nature v.361 Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene Levine, B;Q. Huang;J.T. Isaacs;J.C. Reed;D. Griffin;J.M. Hardwick https://doi.org/10.1038/361739a0
  14. J. Virol. v.72 Antiapoptotic but not antiviral function of human bcl-2 assists establishment of Japanese encephalitis virus persistence in cultured cells Liao, C.L.;Y.L. Lin;S.C. Shen;J.Y. Shen;H.L. Su;Y.L. Huang;S.H. Ma;Y.C. Sun;K.P. Chan;L.K. Chen
  15. J. Virol. v.73 Hepatitis C virus core protein inhibits Fas-and tumor necrosis factor alpha-mediated apoptosis via NR-kappa B activation Marusawa, H.;M. Hijikata;T. Chiba;K. Shimotohno
  16. Br. J. Exp. Pathol. v.69 Immunopathological study of spleen during Japanese encephalitis virus infection in mice Mathur, A.;M. Bharadwaj;R. Kulshreshtha;S. Rawat;A. Jain;U.C. Chaturvedi
  17. J. Virol. v.70 Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype Ray, R.B.;L.M. Lagging;K. Meyer;R. Ray
  18. Fields Virology. Flaviviridae: The viruses and their replication Rice, C.M.;D.M. Knipe(et al.)(eds)
  19. Virology v.229 Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein Ruggieri, A.;T. Harada;Y. Matsuura;T. Miyamura https://doi.org/10.1006/viro.1996.8420
  20. J. Virol. v.69 Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells Sakamuro, D.;T. Furukawa;T. Takegami
  21. DNA Cell Biol. v.18 A G1 cell cycle arrest induced by ligands of the Reovirus typre 3 receptor is secondary to inactivation of p21 and mitogen-activated protein kinase Saragovi, U.H.;N. Rebai;G.M. Di Gugleielmo;R. Macleod;J. Sheng;D.H. Rubin;M.I. Greene https://doi.org/10.1089/104454999314908
  22. J. Virol. v.75 Influenza virus NS1 protein induces apoptosis in cultured cells Schultz-Cherry, S.;N. Dybdahl-Sissoko;G. Neumann;Y. Kawaoka;V.S. Hinshaw https://doi.org/10.1128/JVI.75.17.7875-7881.2001
  23. Curr. Biol. v.6 Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis Sommer, R.J.;P.W. Sternberg https://doi.org/10.1016/S0960-9822(02)00421-9
  24. Science v.265 Potential role of human cytomegalovirus and p53 interaction in coronary restenosis Speir, E.;R. Modali;E.S. Huang;M.B. Leon;F. Shawl;T. Finkel;S.E. Epstein https://doi.org/10.1126/science.8023160
  25. J. Virol. v.71 Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest Stewart, S.A.;B. Poon;J.B. Jowett;I.S. Chen
  26. J. Virol. v.70 The adenovirus death protein (E3-11 .6K) is required at very late stages of infection for effecient cell lysis and release of adenovirus from infected cells Tollefson, A.E.;A. Scaria;T.W. Hermiston;J.S. Ryerse;L.J. Wold;W.S. Wold
  27. J. Virol. v.72 Hepatis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis Zhu, N.;A. Khoshnan;R. Schneider;M. Matsumoto;G. Dennert;C. Ware;M.M. Lai