探討埃及斑蚊訊息肽酶對於登革病毒複製之影響

Effects of mosquito signal peptidases on dengue virus replication in Aedes aegypti

蔡燕儀(Yan-Yi Tsai)

指導教授 ： 蕭信宏

國立臺灣大學/醫學院/微生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903330

摘要

蚊子是許多疾病的傳播媒介，例如：瘧疾、屈躬病、日本腦炎、西尼羅熱、登革熱以及茲卡等，其中，登革熱為非常重要的熱帶疾病之一。根據世界衛生組織統計顯示，全世界每年大約有3.9億的案例，其中約有9600萬的人口具有明顯的臨床症狀。而台灣也是登革熱盛行地區之一，並且在2014及2015年爆發嚴重的疫情，根據統計資料顯示在2014至2015年間，即有超過5萬件的感染案例。雖然目前已有登革熱疫苗上市，但其成效還不是很穩定，對於登革熱病患目前大部分仍採取支持性療法。因此，控制登革熱較實際可行的方法便是透過病媒的控制；要達成此目標，需要對登革病毒與病媒蚊的交互關係有更深入的瞭解。根據先前文獻報導，已知訊息肽酶會參與在蛋白質修飾上，而哺乳類宿主的訊息肽酶對於黃熱病毒屬的病毒複製相當重要，因此我們進一步探討埃及斑蚊的訊息肽酶對於登革病毒複製及傳播的重要性。我們利用RNA干擾的方式將埃及斑蚊的兩個訊息肽酶：SPCS2、SEC11剔除，並且進行西方墨點法、定量即時聚合酶鏈鎖反應和溶斑試驗。結果顯示，SPCS2、SEC11這兩個基因對於登革病毒複製是重要的，並且會影響到登革病毒的效價。另外，在我們的實驗中發現，在埃及斑蚊體內登革病毒會產生兩個不同大小的套膜蛋白片段，不同於過去的報導，哺乳類細胞中登革病毒的套膜蛋白只有一個片段；且在埃及斑蚊的脂肪組織、中腸、卵巢及唾腺都這樣的現象，唯獨在埃及斑蚊的唾液裡僅偵測到大片段的套膜蛋白訊號。我們推測套膜蛋白在埃及斑蚊體內會有進一步的切割，因此我們想探討這樣的切割對於登革病毒複製及傳播的重要性。首先我們利用SignalP進行切割位點預測，發現在套膜蛋白第36個胺基酸上有類似訊息肽酶的切割位點。接下來，我們將登革病毒套膜蛋白上預測的訊息肽酶切割位點進行點突變，再利用Western blot分析，實驗結果顯示經過突變的組別相對於對照組，套膜蛋白較無法被順利切割，因此我們推測套膜蛋白的特定切割位點，可能是訊息肽酶作用的位置。未來我們將將進一步探討SPCS2、SEC11基因對套膜蛋白切割的影響。

關鍵字

埃及斑蚊 ； 登革病毒 ； 訊息肽酶 ； 病毒複製

並列摘要

Mosquitoes act as vectors to carry and spread disease to humans, including malaria, chikungunya, West Nile fever, dengue fever, and Zika. Among these diseases, dengue fever is currently one of the world’s most important tropical diseases. According to the statistics from World Health Organization, 390 million individuals were infected each year, with up to 96 million symptomatic infections. Taiwan is also one of the prevalent areas of dengue fever, and a serious outbreak occurred in 2014 and 2015 with more than 50,000 cases. Although dengue vaccines have been developed recently, their effectiveness and safety remain to be further elucidated. Hence, vector control becomes an alternative strategy for dengue control. Signal peptidases, which have been shown to be required for flavivirus replication that convert secretory and some membrane proteins to their mature forms by cleaving their signal peptides. Absence of signal peptides resulted in reduced yield of Flaviviridae family members. In the dengue mosquito vector Aedes aegypti, several signal peptidases have been identified but their functions were largely unknown. To investigate the role of signal peptidases on dengue virus reproduction in Aedes aegypti, two signal peptidases gene, SPCS2 and SEC11, were silenced by reverse genetic approach. Silencing of SPCS2 and SEC11 resulted in significant reduction of dengue virus replication. Interestingly, we showed that two isoforms of dengue virus envelope protein were exhibited in the mosquito midgut, salivary gland and fat body whereas only one isoform of E protein was detected in the saliva. We speculate that two isoforms may be generated by an additional cleavage of E protein, and this cleavage is important for dengue virus replication or transmission. We therefore generated a point mutation on the predicted cleavage site of the E protein indicating that K36 may be the additional cleavage site of E protein. In the future, we will investigate the effects of signal peptidases in the cleavage of E protein.

並列關鍵字

Aedes aegypti ； Dengue virus ； Signal peptidase ； virus replication

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