Rta蛋白質在Epstein-Barr virus晚期生活史所扮演的角色

Role of Rta in the late stage of Epstein-Barr virus life cycle

黃翔弘(Hsiang-Hung Huang)

指導教授 ： 張麗冠

國立臺灣大學/生命科學院/生化科技學系/博士(2020年)

https://doi.org/10.6342/NTU202000380

摘要

EB病毒(Epstein-Barr virus)為人類最早發現的致癌病毒，具有潛伏期(latent)與溶裂期(lytic)兩種生活史。溶裂期所表現的Rta蛋白質具有活化病毒基因的能力，另外亦能與病毒複製複合體結合，進而協助病毒基因複製。Rta的多功能使之成為EB病毒要產生具有感染力病毒顆粒時最重要的蛋白質之一。透過電子顯微鏡的觀察，本研究發現在病毒生活史晚期，Rta與病毒殼體結合於細胞核內，因此推測Rta在晚期可能具有活化病毒基因以外的能力。在蔗糖梯度的分析中，Rta與釋放的病毒顆粒分布於相同的蔗糖梯度，進一步使用trypsin digestion與de-tegument assay分析出Rta的性質與tegument protein相同，證實Rta為tegument protein。在GST pulldown與免疫沉澱的實驗中發現Rta與殼體蛋白質VCA、BDLF1、BORF1與BFRF3會直接結合，並於後續的螢光顯微鏡分析中發現Rta與BORF1、BFRF3在溶裂期時會重合於細胞核內，顯示Rta為在細胞核內與殼體結合的inner tegument protein。接著，本研究發現殼體蛋白質BORF1的穩定性會受到泛素化修飾(ubiquitination)的調控，而Rta能夠去除BORF1的泛素化修飾，進而穩定BORF1。此為第一個發現Rta與晚期蛋白質的關聯，並證明Rta為tegument protein的研究。此發現顯示Rta在EB病毒生活史晚期與感染時可能具有新的角色，可以更進一步了解EB病毒生活史的發展。

關鍵字

EB病毒 ； Rta ； Tegument proteins ； BORF1 ； 蛋白質穩定性

並列摘要

Epstein-Barr virus is the first identified human oncovirus with the latent and lytic life cycles. Lytic protein Rta activates viral genes and assists viral genome replication by interacting with viral replicating complex. Multifunction of Rta made it one of the most critical protein when EBV is producing infectious virus particles. In an immunogold blot¬ting analysis, we found that Rta associates with virus particles in the nucleus at the late stage of the lytic cycle, demonstrating Rta is more than a transcription and replication activator. Rta also cosediments with EBV virions, and shows similar properties as the EBV tegument protein in trypsin digestion and de-tegument assay, indicating that Rta is a tegument protein. Glutathione S-transferase (GST)-pulldown assay and immunoprecip¬ita¬tion also showed that Rta interacts directly with EBV capsid proteins, including VCA, BDLF1, BFRF3, and BORF1. In an immunofluorescence analysis, Rta colocalizes with BORF1 and BFRF3 in the nucleus during lytic progression, revealing that Rta is an inner tegument protein. Additionally, this study found that BORF1’s ubiquitination regulates its stability, and Rta can reduce the levels of BORF1’s ubiquitination, thus increasing the stability of BORF1. In conclusion, this study finds that Rta is a tegument protein that may improve EBV capsid stability and critical to capsid assembly.

並列關鍵字

Epstein-barr virus ； Rta ； Tegument proteins ； BORF1 ； protein stability

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