人類 BK 多瘤性病毒主要殼體蛋白 VP1 雙硫鍵的特性分析

BK病毒為多瘤性病毒家族的一員。BK病毒首次在1971年被發現，並以首次發現病患命名病毒名稱。BK病毒的感染是無症狀的，且BK病毒會持續的潛伏於腎臟及泌尿道，但在免疫功能低下及免疫功能喪失的病患中，BK病毒會再活化複製並造成腎細胞溶解死亡。BK病毒主要會引起多瘤性腎病變，多瘤性腎病變主要的症狀有慢性間質性腎炎、輸尿管狹窄、出血性膀胱炎及引起腎臟功能障礙等。BK病毒殼體主要由VP1蛋白所組成，每一個病毒殼體由360個VP1（monomer）所組成，每5個VP1分子形成一個次殼體（五聚體）。先前在其它多瘤性病毒，包括JC病毒和SV40病毒的研究中發現，主要殼體蛋白VP1之雙硫鍵對病毒殼體的穩定及組裝扮演重要角色。但至目前為止仍未清楚BKV VP1雙硫鍵在病毒的感染過程中所扮演的角色。本研究中以non-reducing gel，證實BK病毒確實有雙硫鍵存在，並利用質譜儀（LC/MS/MS）鑑定出在BK病毒7個cysteine中，Cys9-Cys9的位置有分子間（inter-chain）雙硫鍵存在。以定位點突變製造出cysteine突變的genome，利用genomic transfection 及infection得到cysteine突變的病毒。再以間接免疫螢光法(Immunofluo rescence assay) 分析早期蛋白LT及Western blot偵測晚期蛋白質VP1的產生。結果發現，C9A、C49A、C87A、C104A、C207A 這幾個突變株無法產生病毒子代。而C254A和C267A兩個突變株可以產生病毒子代。本研究發現雙硫鍵對於BK病毒的生活史扮演著一個不可或缺的角色。

關鍵字

雙硫鍵；人類多瘤性病毒

並列摘要

BKV (BK virus) is a member of the human polyomavirus family. The BKV was first discovered in 1971 from the urine of a renal transplant patient (named initial BKV). Primary infection by BKV is generally asymptomatic. BKV can be latent persistently in kidney and urinary tract. In immunosuppressant and immunoincompetent patients, actively replicating BKV results in lytic disruption of the kidney cells. BKV is the major cause of polyomavirus-associated nephropathy (PVN). The PVN includes chronic interstitial nephritis, ureteral stenosis or hemorrhagic cystitis, which will lead to renal dysfunction. The capsid of BK virus is composed of pentameric capsomeres derived from VP1. The disulfide bonds of VP1 have been reported to play important roles in virion assembly and progeny propagation of JCV and SV40. However, it is not clear about the effects of disulfide bonds of VP1 on viral infection. In this study, the goal is focused on the characteristics of disulfide bonds of VP1 during life cycle of BKV. It revealed that disulfide bonds caused formation of dimers and trimers of VP1 linkages in the virion by non-reducing SDS-PAGE. Inter-chain disulfide linkage between Cys-9 and Cys-9 was identified by tandem MS (mass-spectrometry). Site-directed mutagenesis and transfection showed that the mutants Cys9A,Cys49A,Cys87A,Cys104A,and Cys207A were defect in virion propagation. The results revealed that Cys-9,Cys-49,Cys-87,Cys-104,and Cys-207 were essential for the assembly of infectious virions of BKV.

並列關鍵字

Human polyomavirus BKV ； Disulfide linkage

參考文獻

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人類 BK 多瘤性病毒主要殼體蛋白 VP1 雙硫鍵的特性分析

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