합포체 분석을 위해 R 펩타이드가 결여된 형광 표지 Moloney 마우스레트로바이러스 Molecular Clone 제조

Construction of a Fluorescently Labeled Infectious R Peptide-Less Moloney MLV Molecular Clone for Analysis of Syncytium

  • 이용진 (단국대학교 첨단과학대학 미생물학과) ;
  • 박진우 (단국대학교 첨단과학대학 미생물학과) ;
  • 이규준 (단국대학교 첨단과학대학 미생물학과) ;
  • 배은혜 (단국대학교 첨단과학대학 미생물학과) ;
  • 박성한 (단국대학교 첨단과학대학 미생물학과) ;
  • 임지현 (단국대학교 첨단과학대학 미생물학과) ;
  • 김새로미 (단국대학교 첨단과학대학 미생물학과) ;
  • 정용태 (단국대학교 첨단과학대학 미생물학과)
  • Lee, Yong-Jin (Department of Microbiology and Institute of Basic Science, College of Advanced Science, Dankook University) ;
  • Park, Jin-Woo (Department of Microbiology and Institute of Basic Science, College of Advanced Science, Dankook University) ;
  • Lee, Kyu-Jun (Department of Microbiology and Institute of Basic Science, College of Advanced Science, Dankook University) ;
  • Bae, Eun-Hye (Department of Microbiology and Institute of Basic Science, College of Advanced Science, Dankook University) ;
  • Park, Sung-Han (Department of Microbiology and Institute of Basic Science, College of Advanced Science, Dankook University) ;
  • Lim, Ji-Hyun (Department of Microbiology and Institute of Basic Science, College of Advanced Science, Dankook University) ;
  • Kim, Sae-Ro-Mi (Department of Microbiology and Institute of Basic Science, College of Advanced Science, Dankook University) ;
  • Jung, Yong-Tae (Department of Microbiology and Institute of Basic Science, College of Advanced Science, Dankook University)
  • 투고 : 2009.04.22
  • 심사 : 2009.07.06
  • 발행 : 2009.09.30

초록

레트로바이러스는 바이러스 외막과 숙주세포막의 융합에 의해 세포내로 들어간다. Moloney 마우스레트로바이러스(murine leukemia virus)의 외막 단백질은 표면단백질(SU)과 막단백질(TM)을 포함하는 85 kDa의 전구체로 합성되는데 바이러스의 성숙과정에 막단백질의 카르복시 말단 16개의 아미노산(R-peptide)이 바이러스의 프로티아제에 의해 절단된다. R 펩타이드가 절단된 막단백질은 Moloney 마우스레트로바이러스에 대한 수용체를 가진 NIH3T3 세포주에서 합포체(syncytium)를 형성한다. R 펩타이드가 절단된 막단백질의 합포체 형성 기작 연구를 위해 R 펩타이드가 절단되어 있으며 표면단백질의 PRR (proline rich region) 부위가 EGFP로 삽입되어진 Moloney full length molecular clone을 만들었다. 이 clone은 NIH3T3 세포에서 합포체를 형성하였으며 형광이 세포질과 세포막에서 관찰되었으나 핵은 염색이 되지 않고 검게 보여 신속 정확하게 합포체 관찰이 가능하였다. 흥미롭게도 절단된 막단백질을 가진 비리온이 NIH3T3 세포에서 광학현미경으로 관찰하였을때는 합포체를 형성하였으나 형광현미경에서는 형광이 관찰되지 않아서 비리온이 세포감염 없이 바이러스-세포 융합 방식으로 합포체를 형성한 것으로 생각되었다. 본 연구에서는 형광의 발현 여부로 합포체 형성을 신속 정확하게 관찰할 수 있는 방법을 개발하였으며 R 펩타이드가 절단된 비리온이 세포 감염 없이 세포와 세포 사이의 융합을 매개할 수 있음을 밝혔다.

Retroviruses enter host cells by membrane fusion between the viral Env proteins on the virus membrane and a virus receptor on the cellular membrane. The envelope protein of the ecotropic Moloney murine leukemia virus is synthesized as a gp85 precursor and is proteolytically cleaved into an extracellular surface unit (SU) and the transmembrane protein (TM). The cytoplasmic tail (16 amino acid; R peptide) of the TM protein is further cleaved by the viral protease during virion maturation. Unlike the wild type Env protrin bearing the R peptide, R peptide-truncated Envelope induces syncytia in susceptible cells. To understand the mechanism of R peptidetruncated Env in syncytium formation, R peptide-truncated Env expressing full-length molecular clone containing EGFP in PRR (proline rich region) of Env was constructed. This molecular clone induced syncytia in transfected NIH3T3 cells, fluorescence was detected in the cytoplasm and at the plasma membrane, while the nuclei did not stain and appeared black by fluorescence microscopy. Interestingly, virions with truncated envelope produced from transfected NIH3T3 cells induced syncytia in NIH3T3 cells, but fluorescence was not detected in the same infected cells. It is believed that cell-free viruses direct the fusion of neighboring cells without infection. Our data suggests that use of EGFP-tagged envelope for monitoring syncytium is a sensitive and convenient method. We also found that virion incorporated the R peptide-truncated Env is able to induce the formation of syncytia by fusion from without.

키워드

참고문헌

  1. Albritton, L.M., J.W. Kim, L. Tseng, and J.M. Cunningham. 1993. Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses. J. Virol. 67, 2091-2096
  2. Anderson, K.B. and H. Skov. 1989. Retrovirus-induced cell fusion is enhanced by protease treatment. J. Gen. Virol. 70, 1921-1927 https://doi.org/10.1099/0022-1317-70-7-1921
  3. Chung, M., K. Kizhatil, L.M. Albritton, and G.N. Gaulton. 1999. Induction of syncytia by neuropathogenic murine leukemia viruses depends on receptor density, host cell determinants, and the intrinsic fusion potential of envelope protein. J. Virol. 73, 9377-9385
  4. Eckert, D.M. and P.S. Kim. 2001. Mechanisms of viral membrane fusion and its inhibition. Annu. Rev. Biochem. 70, 777-810 https://doi.org/10.1146/annurev.biochem.70.1.777
  5. Erlwein, O., C.J. Buchholz, and B.S. Schnierle. 2003. The prolinerich region of the ecotropic Moloney murine leukaemia virus envelope protein tolerates the insertion of the green fluorescent protein and allows the generation of replication-competent virus. J.Gen. Virol. 84, 369-373 https://doi.org/10.1099/vir.0.18761-0
  6. Germain, E., V.G. Roulin, J. Qiao, P.O. de Campos Lima, and M. Caruso. 2005. RD114-pseudotyped retroviral vectors kill cancer cells by syncytium formation and enhance the cytotoxic effect of the TK/GCV gene therapy strategy. J. Gene Med. 7, 389-397 https://doi.org/10.1002/jgm.691
  7. Jones, J.S. and R. Risser. 1993. Cell fusion induced by the murine leukemia virus envelope glycoprotein. J. Virol. 67, 67-74
  8. Jung, Y.T. and C.A. Kozak. 2003. Generation of novel syncytiuminducing and host range variants of ecotropic Moloney murine leukemia virus in Mus spicilegus. J. Virol. 77, 5065-5072 https://doi.org/10.1128/JVI.77.9.5065-5072.2003
  9. Jung, Y.T., T. Wu, and C.A. Kozak. 2004. Novel host range and cytopathic variant of ecotropic Friend murine leukemia virus. J. Virol. 78, 12189-12197 https://doi.org/10.1128/JVI.78.22.12189-12197.2004
  10. Katja, S., O. Erlwein, B. Alexandra, and B.S. Schnierle. 2004. Murine leukemia virus (MLV) replication monitored with fluorescent proteins. Virology 1, 14 https://doi.org/10.1186/1743-422X-1-14
  11. Kielian, W.J. and A. Helenius. 1983. Membrane fusion proteins of enveloped animal viruses. Q. Rev. Biophys. 16, 151-195 https://doi.org/10.1017/S0033583500005072
  12. Kim, J.W., E.I. Closs, L.M. Albritton, and J.M. Cunningham. 1991. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature 352, 725-728 https://doi.org/10.1038/352725a0
  13. Kubo, Y., C. Tominaga, H. Yoshii, H. Kamiyama, C. Mitani, H. Amanuma, and N. Yamamoto. 2007. Characterization of R peptide of murine leukemia virus envelope glycoproteins in syncytium formation and entry. Arch. Virol. 152, 2169-2182 https://doi.org/10.1007/s00705-007-1054-6
  14. Kubo, Y. and H. Amanuma. 2003. Mutational analysis of the R peptide cleavage site of Moloney murine leukaemia virus envelope protein. J. Gen. Virol. 84, 2253-2257 https://doi.org/10.1099/vir.0.19126-0
  15. Li, M., Z.N. Li, O.Z. Yao, C.L. Yang, D.A. Steinhauer, and R.W. Compans. 2006. Murine leukemia virus R peptide ingibits influenza virus hemagglutinin-induced memebrane fusion. J. Virol. 80, 6106-6114 https://doi.org/10.1128/JVI.02665-05
  16. Loving, R., K. Li, M. Wallin, M. Sjöberg, and H. Garoff. 2008. RPeptide cleavage potentiates fusion-controlling isomerization of the intersubunit disulfide in Moloney murine leukemia virus Env. J. Virol. 82, 2594-2597 https://doi.org/10.1128/JVI.02039-07
  17. Masuda, M., N. Kakushima, S.G. Wilt, S.K. Ruscetti, P.M. Hoffman, A. Iwamoto, and M. Masuda. 1999. Analysis of receptor usage by ecotropic murine retroviruses, using green fluorescent protein-tagged cationic amino acid transporters. J. Virol. 73, 8623- 8629
  18. Murphy, S.L., M. Chung-Landers, M. Honczarenko, and G.N. Gaulton. 2006. Linkage of reduced receptor affinity and superinfection to pathogenesis of TR1.3 murine leukemia virus. J. Virol. 80, 4601-4609 https://doi.org/10.1128/JVI.80.9.4601-4609.2006
  19. Ou, W. and J. Silver. 2003. Role of a conserved amino-terminal sequence in the ecotropic MLV receptor mCAT1. Virology 308, 101-108 https://doi.org/10.1016/S0042-6822(02)00086-7
  20. Park, B.H., B. Matuschke, E. Lavi, and G.N. Gaulton. 1994. A point mutation in the env gene of a murine leukemia virus induced syncytium formation and neurologic disease. J. Virol. 68, 7516-7524
  21. Pinter, A., T.E. Chen, A. Lowey, N.G. Cortez, and S. Silagi. 1986. Ecotropic murine leukemia virus-induced fusion of murine cells. J. Virol. 57, 1048-1054
  22. Ragheb, J.A. and W.F. Anderson. 1994. pH-Independent murine leukemia virus ecotropic envelope-mediated cell fusion : implications for the role of the R peptide and p12E TM in viral entry. J. Virol. 68, 3220-3231
  23. Rozenberg-Adler, Y., J. Conner, H. Aguilar-Carreno, S. Chakraborti, D.S. Dimitrov, and W.F. Anderson. 2008. Membrane-proximal cytoplasmic domain of Moloney murine leukemia virus envelope tail facilitates fusion. Exp. Mol. Pathol. 84, 18-30 https://doi.org/10.1016/j.yexmp.2007.11.001
  24. Siess, D.C., S.L. Kozak, and D. Kabat. 1996. Exceptional fusogenicity of Chinese hamster ovary cells with murine retroviruses suggests roles for cellular factor(s) and receptor clusters in the membrane fusion process. J. Virol. 70, 3432-3439
  25. Wallin, M., M. Ekstrom, and H. Garoff. 2004. Isomerization of the intersubunit disulphide-bond in Env controls retrovirus fusion. EMBO J. 23, 54-65 https://doi.org/10.1038/sj.emboj.7600012
  26. White, J., M. Kielian, and A. Helenius. 1983. Membrane fusion proteins of enveloped animal viruses. Q. Rev. Biophys. 16, 151-195 https://doi.org/10.1017/S0033583500005072
  27. Wong, P.K.Y., P.H. Yuen, and S.J. Kaufman. 1977. Induction of syncytia by Moloney murine leukemia virus in myoblasts defective in differentiation. J. Virol. 21, 319-327