急性嚴重呼吸道症候群冠狀病毒3a蛋白質與細胞因子Cyr61之結合作用

Abstract

急性嚴重呼吸道症候群(SARS)是一種由新興冠狀病毒所引發的非典型肺炎，自大陸廣東省第一起病例之後，廣泛散播到許多國家。台灣於2003年3月出現第一個指標病例，SARS研究團隊很快地也在感染檢體之非洲綠猴上皮細胞株Vero E6 發現冠狀病毒顆粒。SARS-CoV開放編閱架3a基因可轉譯出一個約30 kDa的蛋白質，稱為p30蛋白質，p30在病毒生活史上的重要性尚不清楚。為了了解p30在病毒複製以及致病機轉上可能扮演的角色，本研究利用GST pull down assay和免疫沉澱法探討與p30有結合作用的細胞因子，發現CNN蛋白質家族中的Cyr61與p30有專一性的結合作用。Cyr61以92-212胺基酸區域與p30進行結合。過去的研究已知，Cyr61是一種分泌性蛋白質，分泌到細胞外的Cyr61會與細胞表面受體結合傳遞下游訊息，例如NF-κB與磷酸化Akt等。為了釐清p30與Cyr61蛋白質之間的結合作用會不會影響Cyr61蛋白質的分泌與下游訊息的傳導，在本研究中將p30表現質體轉染到Vero E6細胞株，兩天後以西方墨點法分析細胞外基質(extracellular matrix)中Cyr61產量和細胞中訊息傳遞因子的表現量。結果發現，在p30轉染表現的細胞中Cyr61蛋白質分泌到細胞外基質的情形沒有受到影響，NF-κB的總蛋白質量亦未有改變，但是磷酸化Akt以及Bax蛋白質量都受到p30蛋白質轉染表現而影響。Bax為Bcl-2 蛋白質家族中的一員，且具有促進細胞凋亡的作用機制，然而在p30 轉染表現的293細胞中，並未偵測到pro-caspase-3被活化的現象。p30是否會影響KF-κB的核分佈情形，轉染p30是否要在較長天數以後才能偵測到細胞凋亡，p30與Cyr61交互作用之意義為何均待進一步的探討。

An outbreak of atypical pneumonia known as severe acute respiratory syndrome (SARS) first emerged from Guangdong Province of China, and subsequently spread to many countries. A novel coronavirus (SARS-CoV) was identified as the causative agent of SARS. In Taiwan, when the first clinical index patient appeared, efforts have been made and the study teams have soon succeeded to grow and amplify the virus particle in Vero E6 cell line. The ORF 3a of viral genome encodes a protein of 30 kilodalton (p30). To understand the possible roles of the SARS-CoV p30 protein involved in the viral multiplication and pathogenesis of SARS, GST pull down assay and immunoprecipitation analysis were applied to examine cellular factors that interact with p30. In this study, Cyr61, a member of CCN family, was identified to specifically interact with the viral p30 through the VWC domain from amino acid residues 92 to 212. Cyr61 is a secreted protein, it interacts with various cell surface receptors to activate downstream molecules, such as NF-κB and phoshporylated Akt. To understand whether the interaction between p30 and Cyr61 proteins would influence the secretion of Cyr61 and the downstream signal transduction, Vero E6 cells were transiently transfected with p30 expression plasmid. The levels of Cyr61 in extracellular matrix and of the downstream molecules were examined by Western blot analysis two days post transfection. The results demonstrated that neither the Cyr61 protein level in extracellular matrix nor the total amount of NF-κB was affected. Nevertheless, the expression patterns of phoshporylated Akt and Bax were changed. Because Bax protein is a member of Bcl-2 family bearing pro-apoptosis function, the possibility of p30 to activate pro-caspase-3 was examined. However, no cleaved caspase-3 activated forms were detected in p30-expressing 293 cells. The biological significances of the interaction between p30 and Cyr61 remain to be elucidated.