EB病毒單股DNA結合蛋白BALF2被包裹入被膜蛋白層的過程之探討

Abstract

EB病毒屬於帶有外套膜的病毒，在其外殼及外套膜之間有一層被膜蛋白。被膜蛋白層的主要成分是一些來自病毒或是細胞的蛋白。被膜蛋白已被證實可以協助成熟病毒顆粒進行下一輪的感染，但是目前被膜蛋白被選擇並包裹進被膜蛋白層的機制還是未知的。先前的研究發現EB病毒單股DNA結合蛋白BALF2以及EB病毒蛋白激酶BGLF4會隨著病毒進入溶裂期晚期而部分分布在細胞質的近核區域中。此外，它們與EB病毒細胞質組裝區域的諸多蛋白質例如BBLF1和GM130有共位的情形。有趣的是，BALF2及BGLF4在病毒溶裂期都是在細胞核中執行功能，但他們最終都會被包裹至病毒的被膜蛋白層中。我們認為這些在細胞核中分布的蛋白可能會藉由與病毒的核外殼緊密相連而隨著核外殼一起被轉移至細胞質，為了探索這個可能性，我們建立了一個純化胞外病毒顆粒的實驗方式。純化出來的病毒顆粒會再藉由介面活性劑來去除外套膜後以0.1 M、0.5 M、1 M的KCl處理，再利用被膜蛋白對於外殼的親和性強弱對其區分。這些樣本以質譜儀的方式分析後來將這些被膜蛋白區分為外層被膜蛋白及內層被膜蛋白。我們想要對被膜蛋白之間的相互作用做深入的探討，先前的研究發現Flag-BALF2和HA-BVRF1可以在HEK-293T細胞中一起被免疫共沉澱下來，並且在NA細胞中BVRF1可以被BGLF4和BFRF1免疫共沉澱下來。因為BVRF1是和黏附於EB病毒外殼的蛋白且BFRF1是和外殼出核相關的蛋白，所以我們猜想BALF2以及BGLF4可能是藉由與BVRF1相互作用而被從細胞核帶到細胞質的。在本研究中，我們用免疫共沉澱法去觀察BALF2和BVRF1之間的交互作用。而為了進一步了解BALF2的哪些功能區域對於它從細胞核移動至細胞質是重要的，我們建構了帶有Flag以及各種功能區域缺失的BALF2質體。我們利用免疫螢光染色法以及免疫共沉澱法來探討這個問題。綜合以上，我們成功建立了純化病毒的方式，而為了對被膜蛋白被包裹的機制有更進一步的了解，被膜蛋白之間是藉由什麼功能區域相互作用的還需再進行確認。

Epstein-Barr virus (EBV) is an envelope virus. A tegument layer sit between its nucleocapsid and envelope. The components of the tegument layer are viral and cellular proteins. Tegument proteins are known to facilitate the second round infection, but how are they be selected and packaged into the tegument layer is still unclear. Previously, we found that EBV single-stranded DNA binding protein BALF2 and EBV protein kinase BGLF4 are partially localized to the cytoplasm juxtanuclear region during the late phase of lytic cycle. Besides, they also co-localized with some proteins contained in cytoplasmic assembly compartment including BBLF1 and GM130. Interestingly, both BALF2 and BGLF4 execute their function in the nucleus during lytic cycle and subsequently packaged into the tegument layer. We hypothesized that these nuclear distributed proteins are closely associated with the nucleocapsids in the nucleus and transported into cytoplasm. To investigate this possibility, we establish a protocol to purify extracellular viral particles. Detergent was used to disrupt the envelope of purified virus then 0.1 M, 0.5 M and 1 M of KCl were used to dissociate tegument proteins with different affinity to the capsids. The samples were then subjected to mass spectrometry to distinguish the inner and outer tegument proteins of EBV. In an attempt to map tegument protein-protein interaction, our previous co-immunoprecipitation data showing that Flag-BALF2 interact with HA-BVRF1 in HEK293T cells, and HA-BVRF1 form immunoprecipitation complex with BGLF4 and BFRF1 in NA cells. Because BVRF1 is a protein that associated with capsid and BFRF1 is a protein involved in nuclear egress, BALF2 and BGLF4 may be “brought” from the nucleus to the cytoplasm by the interaction with BVRF1. In this study, co-immunoprecipitation assay was used to examine the interaction between BALF2 and BVRF1. To map which functional domains are required for BALF2 to translocate from the nucleus to the cytoplasm, plasmids of truncated BALF2 with Flag tag were constructed. Immunofluorescence assay and co-immunoprecipitation assay were used to examine which domains are required for the translocation. Taken together, we established a virus purification protocol. The interaction domain mapping experiments still need to be confirmed for understanding the mechanism of tegumentation process.