登革熱病毒是屬於黃熱病毒科、黃熱病毒屬，主要流行於熱帶以及亞熱帶地區，埃及斑蚊以及白線斑蚊是其主要的中間宿主。登革熱病毒可分為四種血清型，是一種具有單一正股的核醣核酸病毒，主要由三個結構蛋白以及七個非結構蛋白所組成。儘管登革熱病毒的感染會在某些特定的族群發展成為重症，到目前為止仍然沒有有效的抗病毒藥物以及疫苗可以治療。而這些導致某些受試者發生嚴重登革熱的因素仍有待進一步探討，目前為止人類身體營養狀況被認為是可能改變宿主疾病進展的因素之一，因此揭示與宿主營養狀況相關的因素可能為登革病毒感染提供一種潛在的治療策略。鋅是最重要的微量元素之一，人體中的鋅離子不僅調節蛋白質的功能和大量生理過程，同時還參與細胞生長、去氧核醣核酸合成和核醣核酸轉錄等細胞過程的生物學功能。先前的研究表明，鋅離子的恆定可能涉及宿主免疫系統和病毒發病機制，因此我們的目標是想要探討鋅離子與登革病毒感染之間的關係。在本研究中，我們透過使用N, N, N', N'-四（2-吡啶甲基）-1,2-乙二胺 (TPEN)，是一種細胞膜內的鋅離子螯合劑，在人類肝癌細胞 HepG2 細胞中，製造一個缺乏鋅離子的環境，並且感染登革熱病毒觀察影響。在我們的實驗結果顯示出N, N, N', N'-四（2-吡啶甲基）-1,2-乙二胺在預處理或後處理而導致的鋅螯合不會影響 HepG2 細胞存活率，並且可以導致病毒滴度顯著降低。而我們進一步的研究結果顯示，快速同型同步定量偵測系統的實驗結果中病毒核醣核酸的表現確實降低，這表明鋅離子對 HepG2 細胞中登革病毒感染的影響是在核醣核酸複製步驟，而不是病毒的附著及進入。 此外，在 4 小時後將硫酸鋅添加到缺乏鋅離子的 HepG2 細胞中後，發現病毒核醣核酸的表現會恢復，這些結果顯示出鋅離子確實在登革熱病毒中扮演著重要的角色。同時我們透過偵測病毒複製的中間產物雙股核糖核酸來證實鋅離子對於病毒的影響主要在複製階段，並且更進一步發現其影響登革病毒非結構蛋白-5中主要負責病毒核醣核酸複製的核醣核酸依賴性核醣核酸聚合酶。最後，在我們的研究中也發現到螯合掉細胞中的鋅離子並不會直接的影響登革病毒非結構蛋白-5蛋白表現量的改變，但我們發現登革病毒之結構蛋白表現有下降的現象，這可能是病毒在複製的過程中受到影響而導致結構蛋白的合成有問題。這樣的結果顯示出鋅離子並非組成非結構蛋白-5結構中必要之元素，而是扮演著啟動病毒複製開關的一把鑰匙，同時這樣的結果也與先前文獻所提出在非結構蛋白-5中的核醣核酸依賴性核醣核酸聚合酶具有鋅離子結合位相符，由於缺乏鑰匙啟動複製功能進而使下游蛋白表現受到影響。總結以上，在我們的研究中發現到，鋅離子確實在登革病毒的複製中扮演著重要的角色，並且間接的發現鋅離子的缺乏主要會影響到非結構蛋白-5中的核醣核酸依賴性核醣核酸聚合酶，導致複製的的過程受到影響而使下游的結構蛋白合成產生問題。

關鍵字: 登革病毒、鋅離子、N, N, N', N'-四（2-吡啶甲基）-1,2-乙二胺、非結構蛋白-5、核醣核酸依賴性核醣核酸聚合酶、鋅離子結合位

Dengue virus (DENV) is a mosquito-borne flavivirus that is endemic mainly in tropical and sub-tropical countries. The major transmission route of DENV is the vector of Aedes mosquitoes. DENV has four serotypes, and is single positive-stranded RNA virus, encoding three structural proteins and seven non-structure proteins. Although DENV infection induces severe illness in certain affected people, there are no antiviral drugs or effective vaccines for treatment or prevention. The factors contributing to the development of severe dengue in certain subjects remain to be further delineated. Nutritional status has been implicated to be one of contributing host factors that may alter the progression of the disease. Uncovering the factor relevant to host nutritional status may provide a potential therapeutic strategy for DENV infection. Zinc, one of the most important trace elements, not only regulates the functions of proteins and a large number of physiological processes in humans, but also participates in biological functions for cellular processes, including cell growth, DNA synthesis, and RNA transcription. Previous studies have shown that zinc homeostasis may involve in the host immune system, and viral pathogenesis. Hence, we aim to investigate the relationship between zinc and DENV infection. We used N, N, N’, N’- tetrakis (2-pyridinylmethyl)-1,2-ethanediamine (TPEN), a membrane permeable zinc chelator, to perform a zinc depletion environment in HepG2 cells, followed by DENV infection. Results showed that zinc depletion by TPEN, pretreatment or post-treatment, did not affect the viability of HepG2 cells, but led to a substantial reduction in viral titers. Further studies suggested that the level of viral RNA did reduce by qRT-PCR, suggesting that the effect of zinc on DENV infection in HepG2 cells was at RNA replication step rather than the viral entry and internalization. In addition, the level of viral RNA could be restored upon the addition of ZnSO4 to zinc depleted HepG2 cells after 4 hours. To further investigate the mechanism by which zinc affects DENV, we examined the NS5 and NS1 protein expression of DENV because previous studies have mentioned that there was a zinc-binding site on the RdRp of NS5. Unfortunately, our results showed that neither NS5 nor NS1 protein expression was reduced after treating with TPEN in day 1, 2, 3, 5 compared to the DMSO group. Furthermore, zinc treatment only or supplementation after chelating by TPEN did not affect the expression of NS5 and NS1, either. These data suggested that zinc did not influence protein expression of NS5 and NS1 directly. However, we found that the DENV structural protein expressions reduced after treating with TPEN. Collectively, our study had found that zinc ions play an important role in DENV infection, especially on the replication stage of DENV life cycle. The results may provide a new insight of potential therapeutic strategy against the DENV infection.

Key words: Dengue virus, Zn ions, TPEN, viral replication, NS5, RdRp, Zinc binding site

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