探討埃及斑蚊伴侶蛋白BiP對於登革病毒複製與病媒蚊生殖調控之角色

Abstract

登革病毒是由埃及斑蚊所傳播的蚊媒病毒，在全世界造成高度的致病率以及死亡率，為人類重要的致病原之一。由於目前並沒有針對登革病毒的有效的治療方法或是疫苗，病媒蚊控制即成為現今主要預防登革熱的方式。登革病毒在感染到下一個哺乳類動物宿主前，會在埃及斑蚊體內進行複製。其中，登革病毒會與埃及斑蚊體內的蛋白質有交互作用，此現象對於登革病毒在不同階段的發展至關重要。Binding immunoglobulin protein (BiP，又稱GRP78)為一分子伴侶蛋白 (Chaperone)以及調控Unfolded protein response (UPR)的主要調控因子。在先前許多文獻中指出不論在哺乳類或昆蟲中，BiP於眾多黃病毒的生活史中都扮演了相當重要的角色，但對於其如何在埃及斑蚊中調控登革病毒的複製仍是未知的。本篇研究中，我們將目光著重於登革病毒與埃及斑蚊AaBiP在蚊體內的交互作用。根據研究結果，我們發現將AaBiP沉默後會抑制登革病毒的基因體RNA複製、病毒蛋白質以及感染性顆粒的生成，且以餵血經口感染方式的感染率也有明顯抑制的現象。我們也利用免疫共沉澱及螢光標定的方式發現了病毒的非結構性蛋白質-1 (Non-structural protein-1，NS1) 與AaBiP有交互作用。另外，在沉默AaBiP後會促使病毒NS1蛋白質聚集，此結果說明AaBiP可能影響其蛋白質穩定性。此外，我們利用蛋白酶體抑制劑MG132進行實驗；根據實驗結果發現沉默AaBiP並感染了登革病毒的埃及斑蚊在施加了MG132 後，其體內的病毒NS1蛋白質有顯著回升，且呈現劑量依賴的現象。前述的結果使我們認為沉默AaBiP後所導致的蛋白質降解是由蛋白酶體調控。另一方面，我們發現了沉默AaBiP後會抑制埃及斑蚊的生育以及產卵能力，我們進一步發現沉默AaBiP後會導致蚊卵表面構型改變以致無法受精。另外，沉默AaBiP後會抑制卵黃發生 (vitellogenesis) 致使卵巢發育受阻，我們由實驗結果發現其原因為失去AaBiP後抑制了埃及斑蚊vitellogenin蛋白 (AaVg) 與mRNA的表現量，且AaVg蛋白聚集的現象增強。此外，我們也證實了沉默AaBiP後會導致未摺疊蛋白反應的不正常表現，造成細胞自噬的功能異常。綜合以上實驗結果，我們發現了AaBiP在調控登革病毒複製以及埃及斑蚊繁殖能力的雙重角色，而本篇論文也開闢了嶄新且有效進行病媒控制的策略。

The mosquito Aedes aegypti transmits one of the most significant mosquito-borne viruses, dengue virus (DENV), which resulted in marked human morbidity and mortality worldwide. Unfortunately, no specific dengue therapeutics nor effective vaccine is available and prevention is currently limited to vector control. During viral replication in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each stage of the viral cycle. Binding immunoglobulin Protein (BiP, also known as GRP78), a chaperone and master regulator of unfolded protein response (UPR), has been demonstrated to play an essential role in several flavivirus infections. However, the functional role of BiP in regulating dengue virus replication in the mosquito remains largely unknown. In this study, we focus on the interaction between DENV and BiP in the mosquito Aedes aegypti. Our results revealed that silencing of Aedes aegypti BiP (AaBiP) resulted in significant inhibition of DENV viral genome replication, viral protein production, and infectious viral particles biogenesis. In addition, the infection rates after oral infection of dengue virus also showed a significant reduction. Moreover, we also showed that DENV non-structural protein 1 (NS1) interacted with AaBiP by co-immunoprecipitation assay. Additionally, we demonstrated that silencing of AaBiP leads to enhanced DENV NS1 aggregation, indicating that AaBiP mediated viral protein stability. A kinetic study by pulse treatment of MG132, a proteasome inhibitor, in AaBiP-depleted mosquitoes showed that DENV NS1 was drastically elevated. This observation further suggested that AaBiP-mediated viral protein degradation was manipulated by proteasomal machinery. Interestingly, we showed that silencing of AaBiP resulted in inhibition of mosquito fertility and fecundity. We demonstrated that silencing of AaBiP resulted in the collapse of egg micropyle, and further suppressed the fertility of mosquitoes. In addition, depletion of AaBiP inhibited mosquito vitellogenesis due to the reduction of vitellogenin mRNA and elevated aggregation of vitellogenin protein post bloodmeal, further suppressing the development of ovary and mosquito fecundity. Moreover, silencing of AaBiP abnormally turned on the UPR pathway, resulting in the impairment of autolysosome function. Taken together, our results suggested the dual functions of AaBiP in the regulation of dengue virus replication and mosquito reproduction. Information gathered in this study will pave the way toward the establishment of efficient strategies for vector-borne disease control.