Functional Characterization of Phosphorylation of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Nucleocapsid Protein

PRRS 바이러스 Nucleocapsid 단백질 인산화의 기능학적 연구

  • Lee, Chang-Hee (School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University)
  • 이창희 (경북대학교 자연과학대학 생명공학부)
  • Published : 2009.09.28

Abstract

The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a basic multifunctional protein which has been reported to be a serine phosphoprotein with yet-identified functions. As a first step towards understanding the general role of N protein phosphorylation during virus replication, the non-phosphorylated mutant N gene was constructed by mutating all serine residues to alanine. This recombinant N protein was identified to be unphosphorylated, confirming that serine residues truly function as core amino acids responsible for N protein phosphorylation. The PRRSV N protein has been shown to possess the biological features of nuclear localization and N-N homodimerization which individually play critical roles in virus infection. In the present study, therefore, it was attempted to investigate whether these two properties of the N protein are modulated by its phosphorylation status. However, experimental results showed that the non-phosphorylated N protein was still present in the nucleus and nucleolus, and was able to associate with itself by non-covalent interactions. Taken together, the data suggest phosphorylation-independent regulation of N protein nuclear transport or oligomerization, thereby implying the potential involvement of phosphorylation in regulating the activities of the N protein at other levels including RNA-binding capacity.

돼지생식기호흡기증후군 바이러스를 구성하고 있는 뉴클레오캡시드(N) 단백질은 다양한 기능을 가지고 있는 basic 단백질로써 또한 아직까지 밝혀지지 않은 역할을 하는 serine 인산화 단백질로 알려져 있다. 먼저 바이러스가 복제되는 동안 뉴클레오캡시드 단백질 인산화가 어떤 생물학적 역할을 하는지에 대한 이해를 하기 위하여 mutagenesis 방법으로 단백질 내 모든 serine 잔기들을 alanine으로 대체하여 변이 뉴클레오캡시드 단백질을 구축하였다. 이 재조합 뉴클레오캡시드 단백질은 비인산화 단백질로 확인되었고 이는 뉴클레오캡시드 단백질 인산화에 serine 잔기들이 중요한 역할을 한다는 것을 증명하였다. 돼지 생식기호흡기증후군 바이러스 뉴클레오캡시드 단백질은 세포핵 내 이동과 N-N dimer 형성 등의 특이적인 생물학적 특성들을 보유하고 있으며 이들 각각은 바이러스 감염 시 중요한 역할들을 하는 것으로 알려져 있다. 따라서 본 연구에서는 이 두 가지 뉴클레오캡시드 단백질의 특성들이 인산화 여부에 의해 조절되는지 살펴보았다. 하지만 본 연구의 결과들은 비인산화된 뉴클레오캡시드 단백질이 여전히 transfection된 세포의 핵 또는 핵인에서 발현되었고 더욱이 뉴클레오캡시드 자신과 dimer 형성을 할 수 있었다는 것을 보여주었다. 결론적으로 돼지 생식기호흡기증후군 바이러스 뉴클레오캡시드 단백질의 세포핵 내 수송 및 oligomerization 특성들은 인산화 비의존성으로 조절되는 것으로 보여 진다. 아마도 이 인산화 작용은 뉴클레오캡시드 단백질의 RNA-binding 특성등과 같은 다른 수준의 조절과 관련이 있는 것으로 추측되어 진다.

Keywords

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