探討登革病毒非結構性蛋白4A與4B在病毒生活史中的角色;The Study of Roles of Dengue Virus Non-structure 4A and 4B Proteins in Virus Life Cycle

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Title:	探討登革病毒非結構性蛋白4A與4B在病毒生活史中的角色;The Study of Roles of Dengue Virus Non-structure 4A and 4B Proteins in Virus Life Cycle
Authors:	田佳妮;Tian, Jia-Ni
Contributors:	生命科學系
Keywords:	登革病毒;病毒生活史;非結構性蛋白4A;非結構性蛋白4B;病毒複製;內質網壓力蛋白質
Date:	2019-10-29
Issue Date:	2020-01-07 14:25:12 (UTC+8)
Publisher:	國立中央大學
Abstract:	登革熱是一個全球健康問題，影響全世界約39億人。登革病毒分成四種血清型，屬於黃病毒科中的黃病毒屬，登革病毒是由三個結構性蛋白與七個非結構性蛋白組成，非結構性蛋白會形成複製複合物，扮演病毒RNA 複製功能。除了扮演病毒複製功能，對於登革病毒非酵素非結構蛋白4A與4B (NS4A與NS4B) 在生活史中扮演的功能，所知甚少。本研究包括兩個方向，第一篇研究標題是:「針對登革病毒NS4A蛋白進行突變，顯示裸露在細胞質N端的NS4A會影響病毒誘發細胞病變作用」。第二篇研究標題是:「第二型登革病毒NS4B與宿主因子(SERP1)交互作用後，抑制病毒RNA複製，進而減少病毒產量」。第二型登革病毒(DENV-2) NS4A結構包括在裸露在細胞質的N端區域和4個跨膜區域，NS4A主要參與RNA複製和宿主抗病毒反應。然而，對於NS4A蛋白裸露在細胞質N端區域的胺基酸序列，在病毒的生活史中所扮演的角色知之甚少。我們以DENV-2的DNA-launched感染性cDNA與複製子為基礎，將NS4A蛋白N端的胺基酸置換成兩個或三個的丙胺酸，藉此研究NS4A蛋白的功能。在17個病毒突變株中，有9個病毒突變株的RNA複製受到破壞，呈現致死表現型。病毒突變株M2與M14，則呈現病毒產量下降、與野生型病毒相比減少10,000倍，利用複製子分析，病毒突變株M2與M14的病毒複製能力呈現中度受損。藉由分析由M2與M14病毒突變株所衍生的revertant viruses之序列( M2 Rev1與M14 Rev1)，發現皆在NS4A的第21個胺基酸，產生一個由Alanine (A)突變成Valine (V)的突變點 (A21V)。A21V突變點能恢復病毒突變株M2與M14的病毒複製能力，但沒有恢復到類似野生型病毒的RNA複製能力，M2 Rev1與M14 Rev1病毒產量與野生型病毒相比減少100-1,000倍，M2 Rev1與M14 Rev1病毒呈現較小的斑點與類似野生型病毒的組裝與分泌能力。本研究利用MitoCapture staining，測量細胞增生與ATP含量，在HEK-293細胞中發現M2 Rev1與M14 Rev1病毒呈現病毒誘發細胞病變作用功能受損。本研究顯示NS4A蛋白裸露在細胞質的N端區域對病毒複製與病毒誘發細胞病變作用扮演重要角色。宿主感染登革病毒後，會引發細胞觸發內質網壓力，允許病毒複製且不殺死細胞，然而DENV-2蛋白如何和內質網作用，且對登革病毒生命週期產生影響仍未知。本研究利用酵母菌雙雜合系統、次細胞定位、NanoBit互補分析、免疫共沉澱法等研究方法，發現DENV-2 NS4B與內質網壓力蛋白質(SERP1) 交互作用。 Huh7.5 細胞感染第二型登革病毒後，SERP1表現量會增加34.5倍。若在Huh7.5細胞內過度表現SERP1，會抑制病毒產量減少37倍；利用SERP1小髮夾RNA干擾與基因剔除，減少或移除細胞內SERP1表現量，發現病毒產量分別增加3.4倍或16倍。在過度表達SERP1的Huh7.5細胞內轉染DENV-2複製子，發現DENV-2複製能力降低，進而再過度表達NS4B會減輕SERP1抑制DENV-2複製能力，總結上述結果，我們假設在細胞感染病毒後，細胞誘發內質網壓力，SERP1扮演抗病毒角色，抑制病毒感染。本研究提出一個新的抗登革病毒的藥物目標，並促進抗登革病毒的藥物研發。 ;Dengue fever is a global health problem that affects approximately 3.9 billion people worldwide. Dengue virus (DENV) comprises four serotypes (DENV-1,-2,-3, and -4) which belong to the genus flavivirus, family flaviviridae. DENV consists of three structural proteins and seven non-structural proteins. Non-structural proteins form the replication complex responsible for viral RNA synthesis. Besides of role in RNA replication, little is known regarding the role of DENV-2 non-structural proteins, e.g. non-structural protein 4A (NS4A) and 4B (NS4B), during virus life cycle. Two related projects regarding NS4A and NS4B are included in the thesis. The title of first study is “Mutagenesis of the Dengue Virus NS4A Protein Reveals a Novel Cytosolic N-terminal Domain Responsible for Virus-Induced Cytopathic Effects and Intramolecular Interactions within the N-terminus of NS4A”. The title of second study is “A Dengue Virus Type 2 (DENV-2) NS4B-interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication”. The NS4A protein of DENV-2 has a cytosolic N-terminus and four transmembrane domains. NS4A participates in RNA replication and the host antiviral response. However, the roles of amino acid residues within the N-terminus of NS4A during DENV life cycle are not clear. We explored the function of DENV-2 NS4A by introducing a series of alanine substitutions into the N-terminus of NS4A in the context of a DENV-2 infectious clone or subgenomic replicon. Nine of seventeen NS4A mutants displayed a lethal phenotype due to the impairment of RNA replication. M2 and M14 displayed a more than 10,000-fold reduction in viral yields and moderate defects in viral replication by a replicon assay. Sequencing analyses of pseudorevertant viruses derived from M2 and M14 viruses revealed one consensus reversion mutation, A21V, within NS4A. The A21V mutation apparently rescued viral RNA replication in the M2 and M14 mutants although not to wild-type (WT) levels but resulted in 100- and 1,000-fold lower titers than that of the WT, respectively. M2 Rev1 (M2 + A21V) and M14 Rev1 (M14+ A21V) mutants displayed phenotypes of smaller plaque size and WT-like assembly/secretion by a transpackaging assay. A defect in the virus-induced cytopathic effect (CPE) was observed in HEK-293 cells infected with either M2 Rev1 or M14 Rev1 mutant virus by MitoCapture staining, cell proliferation, and ATP levels. In conclusion, the results revealed the essential roles of the N-terminal NS4A in both RNA replication and virus-induced CPE. Intramolecular interactions in the N-terminus of NS4A were implicated. Host cells infected with DENV often trigger endoplasmic reticulum (ER) stress, a key process that allows viral reproduction without killing host cells until the late stage of the virus life cycle. However, little is known regarding which DENV viral proteins interact with ER machinery to support viral replication. We identified and charecterized a novel host factor, stress-associated ER protein 1 (SERP1), that interacts with the DENV-2 NS4B protein by several assays, e.g. yeast two-hybrid, subcellular localization, NanoBiT complementation, and co-immunoprecipitation. A drastic increase (34.5-fold) in SERP1 gene expression was observed in DENV-2-infected or replicon-transfected Huh7.5 cells. SERP1 overexpression inhibited viral yields (37-fold) in DENV-2-infected Huh7.5 cells. In contrast, shRNAi-knockdown and knockout of SERP1 increased viral yields (3.4- and 16-fold, respectively) in DENV-2-infected HEK-293 and Huh7.5 cells, respectively. DENV-2 viral RNA replication was severely reduced in stable SERP1-expressing Huh7.5 cells transfected with DENV-2 replicon plasmids. Overexpression of DENV-2 NS4B alleviated the inhibitory effect of SERP1 on DENV-2 RNA replication. Taking these results together, we hypothesized that SERP1 may serve as an antiviral player during ER stress to restrict DENV-2 infection. Our studies revealed novel anti-DENV drug targets that may facilitate anti-DENV drug discovery.
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