EB病毒BVRF1基因產物之分析

EB病毒的BVRF1蛋白質被推測為是一個可能與外殼連接的被膜蛋白(capsid associated tegument protein)。在BVRF1的同源蛋白UL25的研究中，發現UL25蛋白質在疱疹病毒科的第一型單純疱疹病毒(herpes simplex virus 1, HSV-1)的入殼過程（encapsidation）中是必須的，且主要參與在穩定病毒的DNA-蛋白質殼（nucleocapsid）構造的功能，而不會直接參與病毒DNA截切的過程；然而在與HSV-1屬同一亞科的偽狂犬病病毒（PrV）的UL25對於DNA-蛋白質殼離開核膜（nuclear egress）的過程是重要的，但並不會穩定DNA-蛋白質殼的構造。目前尚無任何研究探討EB病毒BVRF1蛋白質在病毒顆粒形成過程中的角色。由yeast two-hybrid研究中發現BVRF1蛋白質與BFRF1 、BFLF2及BGLF4蛋白質有交互作用。BFRF1 與BFLF2蛋白質是病毒DNA-蛋白質殼通過宿主細胞核膜重要的蛋白質；而BGLF4蛋白質是EB病毒目前已知唯一的蛋白質激酶，且最近的報導顯示BGLF4蛋白質也與DNA-蛋白質殼出核有關，此結果暗示著BVRF1蛋白質可能在病毒顆粒的形成過程中扮演重要的角色。本實驗的目的在瞭解BVRF1蛋白質的基本特性及其可能的生物性功能。首先觀察當NA細胞處理TPA/sodium butyrate 使細胞內的EB病毒進入溶裂期24小時之後，利用BVRF1多株抗體可偵測到BVRF1蛋白質的表現；進一步利用免疫螢光染色分析發現BVRF1蛋白質主要表現在細胞質，然而經由細胞質核分離 (subcellular fractionation)分析，顯示BVRF1蛋白質在細胞內質核的分佈約各佔一半，同時藉由螢光酵素-報導基因檢測發現BVRF1啟動子主要是受到Rta蛋白質所調控。此外，利用共同免疫沉澱法也證明HA-BVRF1與BGLF4蛋白質在細胞內有交互關係；而內源性BFRF1與BGLF4及HA-BVRF1這三個蛋白質亦有交互關係；當在HeLa細胞共同表現BGLF4與HA-BVRF1蛋白質時，在電泳分析下發現HA-BVRF1的分子量有增加，暗示HA-BVRF1可能為BGLF4蛋白激酶之受質。

關鍵字

EB病毒； BVRF1 ；被膜蛋白； BFRF1 ； BFLF2 ； BGLF4

並列摘要

Epstein-Barr virus (EBV) BVRF1 was recognized as a capsid-associated tegument protein through the sequence homology with the Herpes simplex virus type 1 (HSV-1) UL25 gene. It was demonstrated previously that UL25 protein is required for encapsidation but not for cleavage of replicated viral DNA. In addition, the UL25 of pseudorabies virus (PrV) which also belongs to α-herpersvirus family also was shown to play an important role on nuclear export of viral nucleocapsides. The observation from yeast two-hybrid assay indicated that BVRF1 could interact with BGLF4 (protein kinase), BFRF1 and BFLF2. The BFRF1 and BFLF2 proteins were identified to be necessary for efficient viral envelopment at the nuclear membrane. Recent study also indicated that BGLF4 might be involved in the egress of viral nucleocapsids. Taken together with the analogy to the crucial roles of other viral UL25 and findings from yeast two-hybrid assay, we wondered whether BVRF1 might play an important role in the encapsidation of EBV. Despite that, the expression and biological function of BVRF1 have not been characterized. In this study, the expression kinetics and subcellular localization of BVRF1 products were detected in EBV-positive epithelial and B cells upon induction of lytic cycle by using BVRF1-specific mouse antibodies. BVRF1 is expressed in both the nuclei and cytoplasm of EBV-positive NA cells. The promoter activity of BVRF1 is regulated mainly by the immediate-early transcription activator Rta. Furthermore, HA-BVRF1 was shown to interact with endogenous BFRF1 and BGLF4 in EBV positive NA cells undergoing lytic viral replication by co-immunoprecipitation assays. In addition, the higher molecular mass of HA-BVRF1 was observed in SDS-PAGE analysis when BGLF4 was coexpressed in HeLa cells, suggesting HA-BVRF1 could be a potential subtract of BGLF4 protein kinase. Overall, we suggest the BVRF1 may participate in viral encapsidation and egress of viral nucleocapsids.

並列關鍵字

Epstein-Barr virus (EBV) ； BVRF1 ； tegument protein ； BGLF4 ； BFRF1 ； BFLF2 ； nuclear egress ； encapsidation

參考文獻

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Bjerke, S. L. & Roller, R. J. (2006). Roles for herpes simplex virus type 1 UL34 and US3 proteins in disrupting the nuclear lamina during herpes simplex virus type 1 egress. Virology 347, 261-76.

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EB病毒BVRF1基因產物之分析

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