Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Production of the Polyclonal Antibody That Recognizes the Mutant M Protein of Japanese Encephalitis Virus: Role of Its Charged Residues in Virus Production

일본뇌염바이러스의 Mutant M 단백질에 반응하는 다클론항체의 생산: 극성 아미노산 잔기의 바이러스 생산과정에서의 역할

  • Kim, Jeong-Min (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Yun, Sang-Im (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Song, Byung-Hak (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kim, Jin-Kyoung (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Lee, Young-Min (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University)
  • 김정민 (충북대학교 의과대학 미생물학교실, 의학연구소) ;
  • 윤상임 (충북대학교 의과대학 미생물학교실, 의학연구소) ;
  • 송병학 (충북대학교 의과대학 미생물학교실, 의학연구소) ;
  • 김진경 (충북대학교 의과대학 미생물학교실, 의학연구소) ;
  • 이영민 (충북대학교 의과대학 미생물학교실, 의학연구소)
  • Received : 2010.05.10
  • Accepted : 2010.06.01
  • Published : 2010.06.30
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Abstract

Japanese encephalitis virus (JEV), a member of the mosquito-borne flaviviruses, causes epidemics of viral encephalitis in the Southeastern Asia. JEV is a small enveloped virus with a positive-sense RNA genome; the infectious virion consists of three structural proteins, namely capsid, membrane (M; a mature form of its prM precursor), and envelope proteins. Here, we investigated a role of the charged residues found at the N-terminus of the JEV M protein in virus production. Using an infectious JEV cDNA, we generated two mutant cDNAs, Mm1 and Mm2, by charged-to-alanine substitution for $E^9$ and $K^{15}K^{16}E^{17}$ residues of the M protein, respectively. By transfection of wild-type or each of the two mutant RNAs transcribed from the corresponding cDNAs, we found that Mm2, but not Mm1, had a ~3-log decrease in virus production, even though a comparable amount of all three structural proteins were produced in transfected cells. Interestingly, the prM protein expressed in Mm2 RNA-transfected cells was not recognized by the polyclonal antiserum raised against the N-terminal 44 amino acids of the wild type M protein, but reacted to the antiserum raised against the corresponding region of the mutant Mm2. Our results indicate that three charged residues ($K^{15}K^{16}E^{17}$) in JEV M protein play a role in virus production. Two polyclonal antisera specifically recognizing the wild-type or Mm2 version of the M protein would provide a useful reagent for the functional study of this protein in the virus life cycle.

일본뇌염바이러스(Japanese encephalitis virus)는 모기 매개성 플라비바이러스에 속하며, 주로 동남아시아 지역에서 유행성 바이러스성 뇌염을 일으킨다. 일본뇌염바이러스는 외피를 가진 작은 바이러스로서, 양성가닥 RNA 게놈을 가지고 있다. 감염성을 띤 바이러스 입자는 capsid (C), membrane (M; prM 전구체로부터 생성), 및 envelope (E)과 같은 3개의 구조단백질로 이루어져 있다. 본 연구에서는 일본뇌염바이러스 생산과 정에 M 단백질의 N-말단부위에 위치한 극성 아미노산 잔기의 역할을 분석하였다. 일본뇌염바이러스의 infectious cDNA를 활용하여, M 단백질의 $E^9$와 $K^{15}K^{16}E^{17}$ 잔기를 알라닌으로 치환시킨 2개의 mutant cDNA (Mm1과 Mm2)를 각각 제작하였다. 각각의 cDNA로부터 합성된 mutant RNA를 세포에 트랜스펙션시킴으로써, 비록 세포 내에 축적된 3개의 구조단백질양은 변화가 없으나, 이들 세포로부터 생산된 바이러스의 양은 Mm2 RNA의 경우 ~1,000배 감소됨을 관찰하였다. 흥미롭게도, Mm2 RNA로부터 발현된 mutant M 단백질은 wild-type M 단백질을 인지하는 항혈청에는 반응하지 않았으나, mutant M 단백질을 항원으로 제작된 항혈청에는 반응하는 것을 알 수 있었다. 본 실험결과는 일본뇌염바이러스 M 단백질을 구성하는 3개의 극성 아미노산 잔기($K^{15}K^{16}E^{17}$)가 바이러스의 생산과정에 관여한다는 것을 암시한다. 앞으로, wild-type 또는 mutant M 단백질(Mm2)을 인식하는 2개의 항혈청은 이 단백질의 기능연구에 유용한 재료로 사용될 것으로 기대된다.

Keywords

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