人類多瘤性病毒，BK病毒，結構蛋白磷酸化之功能性探討

Functional analysis of phosphorylation of the structural proteins of human polyomavirus, BK virus

陳碧蓮(Chen, Peilain)

指導教授 ： 張德卿

國立中正大學/理學院/化學暨生物化學系/博士(2011年)

摘要

BK病毒(BKV)是屬於人類多瘤性病毒，在腎臟移植病人，BK病毒感染主要造成多瘤性病毒相關腎病變，臨床上表現間質性腎炎，導致腎臟移植失敗。病毒蛋白質之磷酸化作用對其感染性週期扮演重要的角色。我們最近以LC/MS/MS 分析BK病毒結構蛋白 VP1、 VP2 及 VP3 磷酸化的胺基酸，發現在 VP1 的 Ser-80、 Ser-133、Ser-327 ; VP2 之 Ser-223、Ser-248、Ser-254 及 VP3 的 Ser-129 (相當於VP2-Ser-248) 發生磷酸化。本研究我們更加分析磷酸化的功能，in vitro 以 Pro-Q Diamond phosphoprotein 膠體染色 及 in vivo 以 [32P]-orthophosphate做代謝性標定，證實 BKV 結構蛋白的磷酸化作用。利用定位點突變去改變以 LC/MS/MS 鑑定的磷酸化胺基酸，將突變的 BKV 基因組轉染至 Vero 細胞去分析病毒的繁殖，結果發現 VP1-S80A、VP1-S80-133A、VP1-S80-327A、 VP1-S80-133-327A 及 VP2-S254-A 這些突變株經過轉染之後，其早期蛋白，LT，及晚期蛋白，VP1，幾乎不產生；但是其他突變珠的繁殖則類似原始型 BKV。這些結果表示 VP1-S80 及 VP2-S254 的磷酸化作用是BKV繁殖所必要的。

關鍵字

磷酸化作用 ； 多瘤性病毒相關腎病變(PVN) ； BK病毒

並列摘要

BK virus (BKV) is one of the human polyomaviruses. BKV infection may cause of polyomavirus-associated nephropathy (PVN) in patients with renal transplantation. Interstitial nephritis, the major clinical presentation of PVN, may lead to renal graft dysfunction. Phosphorylation of viral proteins may play a crucial role in infectious life cycle of a virus. Recently, the phosphorylated amino acid residues on the structural proteins, VP1, VP2 and VP3, of BKV have been identified by LC-MS/MS in our laboratory. Amino acids Ser-80, Ser-133 and Ser-327 on VP1, Ser-223, Ser-248 and Ser-254 on VP2, and Ser-129 (VP2-Ser-248) on VP3 were phosphorylated. In this study, we further performed functional analysis of the phosphorylations. Phosphorylation on the BKV structural proteins were demonstrated by Pro-Q Diamond phosphoprotein gel staining in vitro and metabolic [32P]-orthophosphate labeling in vivo. Site-directed mutagenesis was performed to replace all phosphorylated amino acids identified by LC-MS/MS. The mutated BKV genomes were transfected into Vero cells for propagation analysis. Results showed that expressions of the early protein, LT, and the late protein, VP1, of mutants VP1-S80A, VP1-S80-133A, VP1-S80-327A, VP1-S80-133-327A, and VP2-S254-A, were abolished after the genomic transfections. However, propagation of other mutants was similar to that of WT BKV. The results may indicate that phosphorylations of Ser-80 on VP1 and Ser-245 on VP2 are crucial for BKV propagation.

並列關鍵字

phosphorylation ； polyomavirus-associated nephropathy (PVN) ； BKV

參考文獻

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