第二型登革病毒外套膜蛋白質C端柄-嵌入區功能之探討

Abstract

登革病毒 (dengue virus)是眾多在熱帶和亞熱帶傳播的病原體之一。登革病毒是屬於黃病毒科的成員之一，共有四種血清型 (DV1~DV4)。登革病毒的外套膜上包含病毒的前驅膜蛋白質及外套膜白質 (prM及E)。E蛋白質在C端有所謂柄-嵌入區 (stem-anchor region)，其中嵌入區即是由兩個穿膜 (transmembrane) 區域構成。此穿膜區域的基本功能是將整個E蛋白質和雙脂質膜連結在一起。根據前人的研究指出對於蜱傳腦炎病毒 (TBEV) 和日本腦炎病毒 (JEV)，只需要表現prM和E蛋白質就足夠產生細胞外似病毒粒子 (extracellular virus-like particles，EPs)。但是對DV2，只有在E蛋白質的柄-嵌入區(即C端20%) 置換成JEV相對序列之下才會有大量的EPs產生；而只置換E蛋白質的嵌入區 (即C端10%) 之下只有產生少量EPs。若全段表現DV2的prM和E蛋白質則得不到大量EPs。所以JEV的柄-嵌入區之序列必定有某些促進EPs產生的因子。 本研究的主要目的是探討登革病毒E蛋白質C端柄-嵌入區對於形成EPs的重要序列或胺基酸。在本研究的第一部分，根據前人之前結果我們利用實驗室已有之全長DV2之prM/E質體 (pCBD2)，帶有JEV嵌入區之prM/E質體 (pCBD2J442) 以及帶有JEV柄-嵌入區之prM/E質體 (pCBD2J396)，進一步建構了帶有JEV柄區的prM/E質體 (pCBD2J396D442)，以及擴充JEV嵌入區之prM/E質體 (pCBD2J430)，來探討柄-嵌入區域對EPs有重要影響之序列。結果顯示，僅有柄區或嵌入區置換成JEV序列，並不足以產生大量EPs。另外擴充JEV嵌入區序列之質體也不足以產生大量EPs，顯示柄區及嵌入區彼此之間可能有交互作用，共同存在之下才足以造成EPs的大量產生。 在本研究的第二部分，我們比較JEV和D2在柄區的胺基酸差異，利用pCBD2作為骨架，選擇其中幾個有明顯不同性質之差異胺基酸做點突變，以探討柄區影響EPs產生的重要胺基酸。結果顯示個別的點突變質體均不足以產生大量的EPs，這與第一部分之結果相符。 黃病毒之prM/E質體可以產生EPs之特性，可用於未來發展疫苗或備製血清學診斷之工具。本研究顯示E蛋白質柄區及嵌入區共同存在之下才能產生大量之EPs。雖未能界定出特定之序列或特定之重要胺基酸，但未來進一步探討柄區及嵌入區之間之交互作用，仍可對如何發展出抑制此重要區域之策略提供重要訊息。

Dengue virus is one of the most important pathogens in the tropical and subtropical areas. Dengue virus belongs to the family Flaviviridae, and has four serotypes (DV1 to DV4). The envelope of dengue virus contains the precursor membrane and envelope proteins (prM/E) encoded by the virus. The E protein belongs to type I membrane protein and has a stem-anchor region at its C-terminal. The anchor region contains two transmembrane segments, which anchors the E protein to the membrane. Previous studies showed that if the entire prM and E protein of tick-born encephalitis virus (TBEV) and Japanese encephalitis virus (JEV) is expressed, the production of extracellular virus-like particles (EPs) is efficient. However, abundant EPs production of DV2 can be achieved, only if the stem-anchor region (C-terminal 20%) of the DV2 E protein is replaced by that of JEV. In the presence of authentic DV2 E protein or if only the anchor region (C-terminal 10%) of DV2 E protein is replaced by that of JEV, EPs production is not efficient. Therefore, the stem-anchor region of JEV sequence contains some enhancing elements for large production of EPs. The main purepose of this study is to investigate the domains or critical amino acids in the stem-anchor region of the DV2 E protein that contribute to the efficient production of EPs. According to the result of previous studies and the constructs available in our lab, including pCBD2 (entire DV2 E protein), pCBD2J396 (containing the stem-anchor region of JEV sequences), and pCBD2J442 (containing the anchor region of JEV sequences), we construct pCBD2J396D442 (containing the stem region of JEV sequences) and pCBD2J430 (containing an extended anchor region of JEV in the first part of our study). In the second part of our study, we use site-directed mutagenesis to generate mutant pCBD2 constructs that contain different amino acid substitutions in the stem region. The ability to generate EPs by these prM/E constructs and mutant pCBD2 constructs were examined. The results revealed that neither prM/E constructs containing a single stem or anchor region of JEV nor mutant pCBD2 constructs can generate EPs efficiently. These findings suggest both the stem and anchor region are important for efficient EPs production. The interaction between the stem and anchor region remains to be investigated in the future. Efficient production of EPs by prM/E construct of flaviviruses has potential to be employed in future vaccine development and in serodiagnosis. Our study revealed that both the stem and anchor regions are important for efficient EPs production. Although no specific motif or amino acid in the stem-anchor region was identified as critical element for EPs production, investigation of interaction between the stem and anchor regions in the future may provide important information for developing strategies to block the function of this region.