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

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研究生：	田佳妮 Jia-Ni Tian
論文名稱：	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
指導教授：	陳盛良岳嶽
口試委員:
學位類別：	博士 Doctor
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2019
畢業學年度：	108
語文別：	英文
論文頁數：	98
中文關鍵詞：	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
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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.

Contents
                                                                       Page 
Declaration    …………………………………………………………………………….        Ⅰ
Publications arising from this thesis…………………………………………………...        Ⅱ
    (A). Referred papers………………………………………………………        Ⅱ
    (B). Abstracts presented in meetings……………………………………..        Ⅱ
中文摘要    …………………………………………………………………………….        Ⅲ
Abstract    …………………………………………………………………………….        Ⅴ
Acknowledgments……………………………………………………………………….        Ⅶ
Table of contents………………………………………………………………………...        Ⅷ
Abbreviation……………………………………………………………………………..        ⅩⅡ
            
Chapter Ⅰ: General Introduction…………………………………………………….        1
    Epidemiology of DENV…………………………………………………..        1
    DENV life cycle…………………………………………………………..        1
    Structure and function of NS4A…………………………………………..        2
    Structure and function of NS4B…………………………………………..        2
    The function of SERP1 in endoplasmic reticulum………………………        4
    DENV mediated endoplasmic reticulum (ER) stress……………………        5
    DENV induced cytopathic effect (CPE)………………………………….        5
    Specific aims of this thesis………………………………………………..        6
Figures    …………………………………………………………………………….        8
Figure I-1    The dengue virus genome………………………………………………...        8
Figure I-2    Schematic diagram of the membrane topology model of DENV-2 NS4A, NS4B, and SERP1 in the ER membrane………………………….……….     8
            
Chapter Ⅱ: Materials and Methods…………………………………………………..        9
    Cell lines and dengue virus strain………………………………………...        9
    Determination of viral yields through plaque-forming assay……….……        9
    Construction of a comprehensive alanine scanning mutagenesis within the N-terminus of NS4A………………………………………………….        9
    Immunofluorescence assay to detect viral antigens………………………        10
    Determination of viral yields through Fluorescent focus assay………..…        11
    Transient replication activity assay of the DNA-launched DENV replicon in BHK21 clone 13 cells………………………………………...        11
    Western blot analysis of viral protein expression………………………...        12
    Selection of pseudorevertants…………………………………….………        13
    Quantification of VLP assembly and secretion using luciferase assay…...        14
    The disruption of mitochondrial transmembrane potential in apoptotic cells were detected by MitoCapture assay………………………………..        14
    Quantification of cell viability by Cell Counting Kit-8 and CellTiter-Glo assays………………………………………………..……………………        15
    Identification of host factors interacting with DENV-2 proteins by a membrane-based split-ubiquitin yeast two-hybrid system………………..        15
    Measurement of SERP1 protein levels in Flag-SERP1-overexpressing Huh7.5 cells by immunoprecipitation analysis…………………………...        17
    Measurement of viral protein levels by immunoprecipitation analysis…..        17
    The localization of NS4B and SERP1 in the endoplasmic reticulum by confocal microscopy……………………………………………………...        18
    NanoBiT complementation assay of SERP1 and NS4B for protein-protein interaction………………………………………………...        18
    Interaction of SERP1 with NS4B by coimmunoprecipitaion (co-IP)…….        19
    Establishment of stable cells expressing shSERP1……………………….        19
    SERP1 knockout cells generated using the type II clustered regularly interspaced short palindromic repeats (CRISPR) system………………...        20
    Measurement of SERP1 RNA levels in knockdown cells and knockout sublines by qRT-PCR……………………………………………………..        21
    Transient replication activity assay of the DNA-launched DENV-2 replicon in Huh7.5 cells…………………………………………………..        22
    Statistical analysis…………………………………………………….…..        22
Figure Ⅱ-1     Construction of DNA-launched DENV2 infectious cDNA and Replicon..        23
Table Ⅱ-1    Primer used for construction of plasmids and sequencing………………..        24
            
Chapter Ⅲ: 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…………………………..    26
Rationale and significance………………………………………………………………..        26
Results    …………………………………………………………………………….        27
    Alanine-scanning mutagenesis in the N-terminus of the DENV2 NS4A protein…………………………………………………………………….        27
    Characterization and phenotypes of NS4A mutant viruses………………        28
            
    Pseudorevertant viruses revealed the compensatory mutation NS4A-A21V, which restored the infectivity and viral titers of the M2 or M14 mutant viruses……………………………………………………….        30
    M2 Rev1 and M14 Rev1 restored RNA replication, although not to the full extent of WT replicon……………………………………….………..        31
    The M2 Rev1 and M14 Rev1 viruses did not apparently interfere with the production of VLPs…………………………………………………...        32
    The M14 Rev1 mutant displayed prominent defects in the virus-induced CPE……………………………………………………………………….        33
Discussion    ……………………………………………………………………………        34
Figures    …………………………………………………………………………….        38
Figure III-1    Schematic diagram of alanine substitution mutations within the N-terminus of NS4A of a DNA-launched DENV2 infectious clone……..        38
Figure III-2    Characterization of the N-terminus of the NS4A WT and mutants………        39
Figure III-3    DAPI-stained images of the WT and NS4A mutations at 7 dpt………….        40
Figure III-4    The reversion mutations rescue the defects of M2 and M14 mutant viruses in virus infectivity, infectious virus production and replication…        41
Figure III-5    Virus-induced CPE was impaired with NS2A NM5, M2 Rev1, or M14 Rev1………………………………………………………………………        43
Figure III-6    The M2, M14, and A21V pseudoreversion mutations located at the predicted membrane topology of the cytosolic N-terminus of NS4A…....        45
Tables    …………………………………………………………………………….        46
Table III-1    Phenotypes of NS4A mutant viruses………………………………..……        47
Table III-2    Summary of the properties of M2 and M14 pseudorevertants……………        45
Table III-3    Summary of the flaviviral N-terminus of NS4A mutants with distinct phenotypes………………………………………………………………..        48
            
Chapter Ⅳ: A Dengue Virus Type 2 (DENV-2) NS4B-interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication…..…    49
Rationale and significance………………………………………………………………..        49
Results    …………………………………………………………………………….        50
    Identification of host factor SERP1 as a DENV-2 NS4B-interacting protein ……………………………………………………………………        50
    SERP1 gene induction by DENV-2 infection and WT replicon transfection………………………………………………………………..        52
    SERP1 has an inhibitory role against DENV-2…………………………..        53
    Viral yields were significantly enhanced in SERP1 knockout cells……...        54
            
    SERP1 overexpression significantly suppresses DENV-2 viral RNA replication…………………………………………………………………        54
    Overexpression of NS4B significantly improves DENV-2 RNA replication in SERP1-overexpressing cells……………………………….        55
Figures    …………………………………………………………………………….        57
Figure Ⅳ-1    Protein-Protein Interactions between SERP1 and NS4B…………………        60
Figure Ⅳ-2    SERP1 expressions were induced in Huh7.5 cells upon DENV-2 infection and WT replicon transfection………………………….…….....        60
Figure Ⅳ-3    SERP1 overexpression inhibited DENV-2 infection, and SERP1 knockdown increased DENV-2 infection………………….……………..        62
Figure Ⅳ-4    Knockout of SERP1 by the CRISPR/Cas9 system in Huh7.5 cells decreased SERP1 mRNA levels, and viral yields were significantly enhanced in SERP1 knockout cells………………………………...………     63
Figure Ⅳ-5    The effect of SERP1 on DENV-2 replicon capacity in Huh7.5 cells…….        64
Figure Ⅳ-6    Overexpression of NS4B improves virus replication in Huh7.5 cells overexpressing SERP1……………………………………………………        65
Figure Ⅳ-7    Hypothetical model of SERP1-mediated DENV-2 infection…………….        66
            67
Chapter Ⅴ: General discussion and future work…………………………………….        68
    Whether NS4A-A21V mutation can rescue the defects of M2 and M14 by restoring the binding of N-terminal NS4A to cell membrane?..............        68
    Whether NS4A-A21V mutation can rescue the defects of M2 and M14 by restoring the NS4A oligomerization?....................................................        68
    Whether overexpression of NS4B alleviates the inhibitory effect of SERP1 on DENV-2 RNA replication by restoring the binding of NS4B to NS2B-NS3 within replication complex?.......................................................    69
    Domain mapping of DENV-2 NS4B and SERP1 interaction…………….        70
Figures    …………………………………………………………………………….        71
Figure Ⅴ-1    The interaction of NS4A WT and mutant peptide with immobilized liposome will be determined using Nicoya liposome sensor chip and SPR..    71
Figure Ⅴ-2    Constructions of the full-length NS4A (1-48) or mutant proteins tagged with the C-terminal fragment of the Flag or EGFP………………………...    71
Figure Ⅴ-3    Schematic diagram of NS2B, NS2B-NS3 and NS4B-HA fusion constructs……………………………………………………………........        72
Figure Ⅴ-3    Schematic representation of SERP1 and NS4B deletion mutant plasmids………………………………………………………………..…        73
References    …………………………………………………………………………….        74

                                

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