EB 病毒的 Rta 蛋白質參與在外鞘組裝的研究

Epstein-Barr virus (EBV)，簡稱 EB 病毒，是人類第四型皰疹病毒，與許多腫瘤疾病相關。當病毒進入溶裂期時，會最先表現極早期蛋白質 (immediate early protein)，Zta 與 Rta，以活化早期蛋白質 (early protein)，協助遺傳物質大量複製，進而活化晚期蛋白質，主要與病毒組裝相關。病毒的外鞘含有主要外鞘蛋白質 (major capsid protein; VCA)，兩種次要外鞘蛋白質 (minor capsid protein; BORF1 and BDLF1)，以及小外鞘蛋白質 (small capsid protein; BFRF3) 等，共同組裝成正二十面體的病毒外鞘，組裝完成後，病毒顆粒釋出 (egress) 感染其它細胞開啟下一個生活史，而外鞘的組裝在晚期是十分重要的一環。目前對於 Rta 的研究著重於極早期以及早期的調控，但在晚期的角色仍不清楚。本篇研究主要探討 Rta 與外鞘蛋白質之間的關係。首先發現，Rta 會與 BORF1 以及 BDLF1 在細胞內結合，並利用 GST-pull down 證明，Rta 能與 BORF1、BDLF1、VCA 以及 BFRF3 等外鞘蛋白質直接結合。接著，以免疫螢光法觀察到 Rta 在溶裂晚期會與 BORF1、 BDLF1，以及 BFRF3 等外鞘蛋白質共同分布於細胞核內。並以蔗糖梯度分離、脫殼實驗以及電子顯微鏡的觀察，證實 Rta 會做為鞘間蛋白質存在於病毒顆粒中。最後，透過變性免疫沈澱法發現 Rta 會降低 BORF1 的去泛素化修飾，穩定外鞘蛋白質，另一方面，藉由冷光報導基因分析，觀察到 BORF1 會降低 Rta 活化早期基因的能力。本研究揭露了極早期蛋白質 Rta 會與外鞘蛋白質會相互結合，且首度提出 Rta 是 EB 病毒的鞘間蛋白質，會提升外鞘蛋白質的穩定性，並受外鞘蛋白質調控 Rta 活化早期基因。

關鍵字

Epstein- Barr Virus (EB 病毒) ； Rta 蛋白質；外鞘蛋白質；病毒顆粒；鞘間蛋白質

並列摘要

Epstein-Barr virus (EBV) is also called human herpesvirus 4 (HHV-4), which is associated with many neoplastic diseases. When the virus enters to lytic stage, it expresses two immediate early (IE) proteins, Rta and Zta, which are required to promote the transcription of early genes, to help the replication of viral DNA. Then, the late proteins are activated, which are required for nucleocapsid assembly and egress. The icosahedron capsid of EBV contains major capsid protein, VCA, two minor capsid proteins, BORF1 and BDLF1, and small capsid protein, BFRF3. After virion assembly, they egress to infect other cells and continue the next life cycle. To date, the functional analysis of Rta focuses on the regulation during the immediate early stage and early stage. However, the role of Rta in the late stage is still unclear. The purpose of this study is to elucidate the relationship between Rta and capsid proteins. First, coimmunoprecipitation assay reveals that Rta colocalizes with BORF1, and BDLF1. And GST pull down assay shows that Rta interacts directly with BORF1, BDLF1, VCA, and BFRF3. Moreover, immunofluorescence reveals that Rta colocalizes with three capsid proteins, including BORF1, BDLF1, and BFRF3 during the late stage of the lytic cycle. Furthermore, Rta is also present in the virions and served as a tegument protein. Finally, denature immunoprecipetation reveals Rta decreesed the ubiquitination of BORF1, stabilizing BORF1; luciferase assay provides BORF1 decreased the transactivation of early genes by Rta. Taken together, this study demonstrated that Rta interacts with EBV capsid, exists in the virions as a tegument protein, increases the stability of capsid proteins, and the activation of erly genes by Rta is regulated by capsid proteins.

並列關鍵字

Epstein- Barr Virus ； Rta ； capsid proteins ； tegument proteins

參考文獻

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EB 病毒的 Rta 蛋白質參與在外鞘組裝的研究

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