BFLF2蛋白之進核及與BFRF1交互作用序列對於EB病毒顆粒釋出之影響

Abstract

屬於γ-皰疹病毒科 (γ-herpesviridae) 的EB病毒 (Epstein-Barr virus) 在細胞核內完成病毒DNA的複製後，其複製完成的DNA會被包裹形成核殼體(nucelocapsid)，當核殼體完成組裝後，會透過出核複合體幫助病毒核殼體離開細胞核進到細胞質，以便進行最後階段的外套膜組裝。EB病毒的出核複合體由BFLF2及BFRF1所組成，分別是單純皰疹病毒 (Herpes simplex virus-1) 的UL31, UL34同源物。目前已知BFLF2單獨表現時會位在細胞質，但當其與BFRF1共同表現時，BFLF2則與BFRF1共位在核膜及細胞質，並且有泡狀產生。因此我們要探討BFLF2及BFRF1之間如何互相調節來幫助病毒核殼體出核，本篇研究著重於探討BFLF2不同的功能區段 (functional domain) 對於病毒複製的影響。先前研究顯示BFLF2胺基酸序列2-102對其進核 (nuclear localization) 及與BFRF1交互作用有重要影響，而本篇研究針對此段胺基酸進行分段序列刪除。我們發現BFLF2胺基酸序列81-107為其與BFRF1的交互作用序列，並且發現BFLF2的進核序列 (nuclear localization signal) 是由三個重要的胺基酸 (47 Arginine, 50 Lysine, 52 Arginine) 及一段較為分散、帶有正電的胺基酸序列所組成，並且藉由輸入蛋白β (importin β) 幫助進核。另外，在帶有EB病毒基因組並剔除BFLF2的細胞 (293TetER/p2089ΔBFLF2) ，發現病毒的分泌程度會因為缺少BFLF2而下降，一旦回補BFLF2便會回復病毒分泌程度。也發現當缺少BFLF2進核序列以及與BFRF1的交互作用序列，病毒的分泌也會受到減縮。綜合結果而論，本篇研究發現了BFLF2的進核序列及其與BFRF1交互作用之序列，而這兩段序列對於病毒分泌有重要的影響。

Epstein-Barr virus is a human gamma herpes virus, which replicates its genomic DNA in the nucleus. Replicated viral DNA is packaged into procapsids as nucleocapsids before the transport from the nucleus into the cytoplasm for subsequent maturation. This nuclear egress process is facilitated by the function of viral nuclear egress complex (NEC) which consisted of BFLF2 and BFRF1, the homologs of UL31 and UL34 in HSV-1, respectively. It was known that BFLF2 localized in the nucleus when expressed alone. However, the distribution of BFLF2 changes to both nuclear rim and cytoplasm with vesicle formation once it was co-expressed with BFRF1. We were intrigued by the different functional domains of BFLF2 in EBV replication. Our previous study indicated that BFLF2 a.a. 2-102 was required for the nuclear targeting and the interaction with BFRF1. To know exact functional motifs of BFLF2 within a.a. 2-102, systemic deletion mutants of BFLF2 were generated. We found that a.a. 81-107 of BFLF2 was responsible for the interaction with BFRF1. In addition, non-canonical nuclear localization signal (NLS) of BFLF2, which consisted of three crucial amino acids (47R, 50K, 52R) and several separated arginines were identified. Although the nuclear localization of BFLF2 is still importin β-dependent, BFLF2 NLS is different from other conserved NLSs of α- and β-herpesviruses. Furthermore, virion secretion was diminished in BFLF2 knock out 293TetER/p2089 EBV bacmid cells, but it was rescued after BFLF2 trans-complementation. In addition, the absence of identified NLS or BFRF1-interacting domain of BFLF2 leads to a decrease on virion secretion. Altogether, these results revealed the nuclear targeting and BFRF1-interacting domains of BFLF2, which are important for virion secretion.