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標題: 典型豬瘟病毒NS2蛋白在細胞中分布情形之研究
Studies on the cellular localization of nonstructural protein 2 of Classical swine fever virus
作者: 田卉萱
Tien, Hui-Hsuan
關鍵字: CSFV;豬瘟病毒;NS2;非結構蛋白2
出版社: 生物科技學研究所
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豬瘟病毒(Classical swine fever virus)又稱豬霍亂病毒(Hog cholera virus),和廣為人知的C型肝炎病毒(Hepatitis C virus)同隸屬於黃病毒科,和牛病毒性下痢病毒(Bovine viral diarrhea virus;BVDV)同隸屬於瘟病毒屬。豬隻感染CSFV後會造成食慾不振、精神萎靡、高熱以及臟器出血等症狀,為一廣泛性的傳染性疾病,多數國家對於感染豬瘟的豬隻採取全面撲殺之政策,嚴重影響經濟效益,為了尋求有效之解決辦法,豬瘟病毒的複製機制被廣泛的探討。典型豬瘟病毒只有一個開放性讀碼區(Open reading frame),可以轉譯出由3,898個胺基酸組成的聚胜肽鏈,然後再進一步截切成12個較小的蛋白質,各司其職的扮演著不同的角色。前人文獻指出,在BVDV寄主細胞中Jiv蛋白(J-domain protein interacting with viral protein)與非結構蛋白2 (NS2)的結合,會誘導NS2蛋白產生自我剪切的活性,使非結構蛋白3 (NS3)自NS2/3分離,進一步與非結構蛋白4A (NS4A)結合,進行下游蛋白的截切並聚合成為RNA複製子,行RNA複製作用。然而在豬瘟病毒的生活史裡,NS2是否如牛瘟病毒一樣會影響RNA複製,或是還有其它的功能?因此我們將由NS2蛋白在細胞中的分佈情形著手,研究NS2在CSFV 的感染過程中可能扮演著什麼樣的功能。本實驗藉由PCR的方式將NS2以及NS2/3接上T7 tag,FLAG tag或EGFP,使用Lipofectamine 2000當作載體,將NS2及NS2/3基因轉染進豬腎細胞中(PK-15),培養24小時後利用免疫螢光染色分析NS2蛋白在豬腎細胞中的表現。初步實驗結果顯示,NS2蛋白可能坐落於內質網上。另一方面,經由TMHMM sever 2.0軟體預測在CSFV NS2蛋白的N端有五個穿膜區塊,可能與NS2蛋白結合於內質網的功能有關,為了驗證這個假設,本實驗將NS2的五個穿膜區塊剔除並於N端接上EGFP tag轉染進豬腎細胞中,培養24小時後利用共軛焦螢光顯微鏡分析,其結果顯示NS2蛋白分佈於整顆細胞中,不再座落於內質網,這表示NS2的五個穿膜區塊對與內質網的結合非常重要。Jiv蛋白也被報導於內質網膜或內質網內腔(lumen)上表現,推測NS2與Jiv蛋白可能以內質網為工作平台進行交互作用,然而NS2蛋白在細胞中之表現非常微弱,推測和其它黃病毒科的NS2蛋白一樣在細胞中有被水解之可能。未來實驗將分為兩部分:自豬的血液細胞中clone出Jiv90基因,研究其與NS2交互作用;另一方面,在細胞培養期間加入蛋白分解體抑制物(MG132),然後再計算NS2蛋白在細胞內的累積量以觀察其消長情形。

Classical swine fever virus (CSFV) the causative agent of swine fever belongs to the pestivirus genus of the family Flaviviridae. Clinical symptoms include anorexia, depression, hyperpyrexia and hemorrhage. Most of the countries in which this disease breaks choose to exterminate all the pigs, leading to huge economic losses. The single strand positive sense CSFV RNA genome contains one single open reading frame that encodes a 3,983 polypeptide which is processed to 12 different proteins. Previous studies indicate that, Jiv protein (J-domain protein interacting with viral protein) from BVDV infected cells acts as a regulating cofactor for the nonstructural protein 2 (NS2) autoprotease and initiates NS2-3 cleavage in trans releasing nonstructural protein 3 (NS3), one of the essential components of the viral RNA replicase complex. Recent studies show that after NS3 release the NS2 still binds to Jiv and prevents further rounds of cofactor function which; an event thought to be the switch to stop RNA replication and start packaging. Though NS2 influences CSFV RNA replication it does not participate in it directly. Whether NS2 involved in other functions of CSFV life cycle is still unknown. Therefore, the distribution of NS2 in porcine kidney cells (PK-15) could be the starting point in understanding its role in CSFV life cycle. In order to pursue localization studies NS2 and uncleavable NS2/3 were fused with T7 and FLAG tags on their N- and C-terminus, respectively. Constructs were transfected using Lipofectamine 2000 in PK-15 cells and the distribution of NS2 was examined 24 h post-transfection using ImmunoFluorescence Assay (IFA) and confocal imaging. Preliminary results using EGFP-ER marker showed that NS2 co-localizes with endoplasmic reticulum (ER). Interestingly this localization corresponds to a cellular host factor Jiv that binds to and influences pestiviral NS2 as mentioned in previous studies. However, the amount of NS2 expressed in cells is low and reports on Hepatitis C Virus (HCV) NS2 suggest that NS2 degrades rapidly in infected cells. To investigate further, we will focus on the distribution of NS2 protein in PK-15 cells using different NS2 mutants to determine the ER localization signal and the putative Jiv binding domain.
其他識別: U0005-0202201016512800
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