Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Construction of a Fluorescently Labeled Infectious R Peptide-Less Moloney MLV Molecular Clone for Analysis of Syncytium

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

  • 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)
  • 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)
  • Received : 2009.04.22
  • Accepted : 2009.07.06
  • Published : 2009.09.30
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Abstract

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.

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.

Keywords

References

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