Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96407
標題: 雞傳染性貧血病毒蛋白特性之研究:VP1與DNA結合能力及VP2與VP3交互作用
Characterization of Chicken Anemia Virus Viral Proteins:DNA Binding Activity of VP1 and Interaction between VP2 and VP3
作者: 賴冠樺
Guan-Hua Lai
關鍵字: 雞貧血病毒;病毒蛋白VP1、VP2、VP3;DNA結合能力;DNA構形喜好性;細胞凋亡;蛋白-蛋白交互作用;Chicken anemia virus;Viral proteins VP1, VP2 and VP3;DNA-binding ability;DNA conformational preference;Apoptosis;Protein-protein interaction
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摘要: 
雞傳染性貧血病(Chicken infectious anemia;CIA)是由雞傳染性貧血病病毒(Chicken anemia virus;CAV)所引起的雛雞再生障礙性貧血和全身性淋巴組織萎縮的傳染病,為養雞業者最常遭遇到的病毒性疾病之一。CAV 病毒可表現三種各具不同功能的蛋白,分別為VP1、VP2 以及VP3。
於本研究中,我們針對CAV病毒可表現之三種蛋白進行不同的特性化分析。首先利用agarose gel shift方法,我們檢驗重組VP1蛋白的DNA結合能力,發現VP1蛋白能結合DNA分子,且此結合與DNA序列無關。同時藉由相同實驗證明重組蛋白VP1亦能與不同構形之DNA分子,如:環狀雙股DNA、線狀雙股DNA、環狀單股DNA與線狀單股DNA等產生交互作用,顯示VP1蛋白的無DNA構形專一性的DNA結合能力。進一步,透過觀察特定構形DNA與不同濃度的重組蛋白VP1間的DNA遲滯訊號差異,我們發現儘管VP1蛋白可與不同構形的DNA交互作用,然而蛋白-DNA結合效益會受到其對特定DNA構形喜好性的影響,顯示VP1蛋白具有偏好結合於特定構形DNA的特性。
VP2蛋白與VP3蛋白皆為CAV的非結構蛋白,兩蛋白可於病毒感染宿主細胞後率先被表現,顯示兩蛋白與CAV病毒複製的重要性。本研究透過螢光影像定位,以及雙分子螢光互補法(Bimolecular fluorescence complementation assay, BiFC)與GST pull-down等蛋白-蛋白交互作用研究方法,證實VP2與VP3蛋白間存在細胞核內的直接性交互作用。我們進一步觀察兩蛋白之交互作用對宿主細胞生理活性之影響。於MDCC-MSB1病毒易感細胞中,共同表現VP2及VP3蛋白時,相較於單獨表現單一病毒蛋白,其細胞凋亡率呈現部份抑制的現象。藉由西方點墨法,我們發現VP3蛋白Thr108位置磷酸化程度明顯下降,推測VP2蛋白抑制VP3蛋白促細胞凋亡活性,乃透過其去磷酸酶活性減少VP3上Thr108位的磷酸化所致。然而,我們亦發現Thr108位置突變後的VP3蛋白,仍保有部份誘導細胞凋亡能力,此結果顯示Thr108之磷酸化具有調節VP3蛋白致凋亡能力,但並非是唯一方式。
綜合上述對CAV病毒蛋白之特性化檢驗成果,本研究為後續探討雞貧血病毒相關議題提供了初步的實驗佐證,相信對於瞭解各病毒蛋白於CAV病毒生活史中所扮演的角色將有莫大的助益。

Chicken anemia virus (CAV) is a worldwide spreading pathogen which is the major cause of Chicken infectious anemia (CIAV) inducing chicken immunosuppressive disease in developed chicken industry of most countries. The CAV genome encoded the sole structural protein VP1, two non-structure proteins VP2 and VP3. In this study, the functional characteristics of three CAV viral proteins were systematically analyzed by different biological methods.
Firstly, the DNA-binding ability of VP1 was determined by using the purified recombinant VP1 protein with various plasmids through detecting DNA fragments retardation after agarose gel shift. The results showed no influence on DNA migration regardless of whole viral genome involved in experimental plasmids indicating VP1 protein has the sequence-nonspecific DNA-binding activity. The interaction between recombinant VP1 protein and difference conformation of DNA molecules such as circular dsDNA, linear dsDNA, circular ssDNA and linear ssDNA were also investigated by the same experiments, and the results also presented the DNA-binding ability of VP1 with tested DNA molecules. Furthermore, the DNA conformational preferences of VP1 protein were analyzed through quantifying the required amounts of VP1 protein for binding to certain conformation of DNA. In order of preferences determined respectively by the binding capacity of VP1 protein were circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular dsDNA and linear dsDNA. The DNA conformational preference characterization of VP1 might endows the protein with extra possible bio-functional roles in CAV replication.
In the next part of this study, the experiment platform established previously were used for detecting the localization of VP2 and VP3 in terms of the direct fluorescent observation using fluorescence microscopy the cell respectively. The presented subcellular co-localization of VP2 and VP3 indicated the protein interaction might existed between the two proteins. Moreover, the reliability of VP2-VP3 interaction were confirmed by using bimolecular fluorescence complementation assay (BiFC) under ex vivo condition and in vitro GST pull-down assay. The results demonstrated that VP2 interacts with VP3 directly and the interaction was mainly in nucleus. Furthermore, when VP2 and VP3 were co-expressed in CAV susceptible cells MDCC-MSB1, the ratio of cell apoptosis was declined significantly compared to sole expressed VP3 gene without VP2 in cells. In addition, the threonine 108 (Thr108) phosphorylation state of VP3 was decreased when co-transfected with VP2 into the cells. These results indicated that VP2 as down-regulator by altering phosphorylation status of VP3 through dual-phosphatase activity to reduce the VP3's apoptosis-inducing ability. However, the unchanged nucleus distribution but partial apoptosis suppression in MDCC-MSB1 after expressing the T108A-mutated VP3 revealed that the Thr108 phosphorylation is not the only way to mediate the pro-apoptosis activity of VP3.
These experimental results above provided the useful information for further research issues such as CAV replication and pathogenicity mechanism to understand the true appearance of CAV life cycle.
URI: http://hdl.handle.net/11455/96407
Rights: 同意授權瀏覽/列印電子全文服務,2020-08-01起公開。
Appears in Collections:生物科技學研究所

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