Rab1A在EB病毒成熟過程中的角色以及其與EB病毒 單股DNA結合蛋白BALF2交互作用關係之探討

Abstract

EB病毒屬於帶有外套膜的皰疹病毒家族，其外套膜與外殼的中間夾層帶有一層被膜蛋白，在細胞質中會被組裝到病毒顆粒內部。被膜蛋白已經被證實可以協助諸多皰疹病毒家族的成熟病毒顆粒有效去進行下一輪的感染，但是其中的組成蛋白是如何精確的篩選以及包裹之過程仍然有許多的未知。在我們先前的研究當中，我們發現原本主要存在於細胞核的EB病毒單股DNA結合蛋白BALF2會隨著病毒進入溶裂期而有在細胞質中的訊號逐漸增加的趨勢，並且與EB病毒細胞質組裝區域的諸多蛋白質有共位的情形，這個現象也與BALF2是一個被膜蛋白的成員需要在細胞質進行組裝可互相應證。為了要進一步去探討被膜蛋白在細胞質中組裝的過程要素，我們利用質譜分析的結果發現，有一群與宿主運輸路徑相關的蛋白與BALF2有交互作用，當中包含了參與在內質網與高基式體之間運輸細胞物質的Rab1A GTPase。在利用共軛焦顯微鏡的實驗結果中，我們首先發現了在EB病毒活化的NA細胞，BALF2會與內生性Rab1A 在細胞質核凹陷處的組裝位點有共位的情形，除此之外，BALF2也可以與GFP-Rab1A一起被免疫共同沉澱下來。當在細胞內表現GTPase活性失活的Rab1A不只會造成BALF2在細胞質組裝區域的訊號降低，其他像是被膜蛋白BBLF1以及高基式體標記蛋白GM130也都會因此而降低在細胞質組裝區域的訊號表現，顯示著Rab1A的正常活性對於病毒細胞質組裝區域形成的重要性。EB病毒成熟之前在細胞質的組裝區域除了被膜蛋白的選擇性包裹以外，醣蛋白的組裝是另一個主要過程。而影像結果顯示當在細胞內表現GTPase活性失活的Rab1A也會造成聚集在細胞質組裝區域聚集的醣蛋白gp350/220之擴散。進一步利用即時連鎖聚合酶的反應定量病毒基因量證實，GTPase活性失活的Rab1A表現會造成成熟病毒的釋出量降低。而當利用RNA干擾技術去去除細胞內Rab1A的表現之後，病毒總體的釋出量也有顯著的下降。綜合以上，Rab1A調控了被膜蛋白在細胞質組裝區域的聚集現象，並且扮演著對病毒的正確釋出一個重要的角色。

Epstein-Bar virus (EBV) is a Herpesviridae enveloped virus that contains a tegument layer between its nucleocapsid and envelope. Tegument proteins, which are known for facilitating next round of infection, are still not well understood for their roles in virus assembly and packaging process. Previously, we found that EBV single-stranded DNA binding protein BALF2 partially localized to the cytoplasm after entering lytic cycle. It is reasonable since BALF2 is a component of the EBV tegument layer. What is more, BALF2 also co-localized with some cytoplasmic assembly compartment containing proteins (BBLF1, BGLF4, GM130). To explore the tegumentation process, a mass spectrometry analysis was produced, and a group of host trafficking pathway related proteins were identified to interact with BALF2, including small GTPase Rab1A. Rab1A is known to function in the cargo delivering process between ER and Golgi. We demonstrated that BALF2 partially co-localized with endogenous Rab1A in the assembly compartment after reactivation using confocal microscope analysis, and BALF2 could also form immunoprecipitation complex with GFP-Rab1A in reactivated EBV-positive NA cell lysates. In addition, by expressing a GTPase activity dominant negative GFP-Rab1A (N124I), the clustering of Rab1A and BALF2 in the cytoplasm was diminished, so did the distribution of other tegument protein (BBLF1) and assembly compartment trans-golgi marker (GM130), suggesting functional Rab1A is required for the formation of cytoplasmic assembly compartment. The importance of Rab1A GTPase activity was further demonstrated in the localization of glycoprotein gp350 at the assembly compartment, which is another major event other than tegumentation that happens at the assembly compartment for virion assembly. Extracellular virion secretion was downregulated when GFP-Rab1A(N124I) was overexpressed. When siRNA was used for Rab1A knockdown, the virion secretion outcome was also downregulated significantly. Taken together, Rab1A regulates the accumulation of EBV tegument proteins at the assembly compartment, and its intact GTPase activity is required for proper virion secretion.