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標題: Regulation of Avian Reovirus Replication and Cleavage of Sigma a by Ubiquitin-Proteasome System and Cleavage of sigma A
Ubiquitin-Proteasome System調控家禽里奧病毒複製及sigma A蛋白切割之研究
作者: 劉宏仁
關鍵字: 生物技術, 畜牧獸醫類;基礎研究
Avian reovirus (ARV) causes several disease syndromes in poultry includingarthritis, malabsorption syndrome, and chronic respiratory disease that result in majoreconomic losses. We have previously demonstrated that σC is an apoptosis inducerand further provided the evidence of ARV-induced apoptosis through activation ofSrc kinase and p53-mitochodrial pathway and then activation of caspases to induceapoptosis. Therefore, the first part of this study is aimed at elucidating whether ARVutilizes the ubiquitin-proteasome system (UPS) for its own benefit and othermechanisms regulation for cells. In our preliminary data we found that proteasomeinhibitor MG132 could reduce ARV-induced cytopathic effect, virus titer, viral proteinexpression and apoptosis. These new findings suggested that ARV-induced apoptosis,signal transduction, viral transcription and translation, viral protein cleavage, and viralreplication may be regulated by UPS.Recently, the presence of σA in nuclei was confirmed in our laboratory byimmunofluorescence antibody assay (IFA) using a monoclonal antibody against ARVσA protein, suggesting that this protein may posses the other biological functions.RNA interference (RNAi) was used to suppress protein expression of the S-classgenome segments of ARV. We discovered for the first time the cleavage of the innercapsid protein σA into smaller fragments in both vero and BHK-21 cells, namely σACand σAN. The amount of fragment σAC could be reduced after suppression of σA byRNA interference. The σA protein is different from other proteins containing signalpeptides since the predicted a cutting site on σA protein is a little far away from bothN- and C-terminal. Previous studies had also reported that localization and functionof a few viral proteins would be affected after being cleaved by proteases linked tocaspases. It was proved that ARV σA protein has no posttranslational modificationlike glycosylation and never enters endoplasmic reticulum. To further elucidate theeffects of cleavage and nuclear translocation of σA on influences on viral assemblyand cell function, therefore, the second part of this project is to study whether themechanism of σA cleavage is related to apoptosis or UPS and the event may becaused directly by caspases or indirectly by proteases from leaky organelles. Inconclusion, this study including two major research points is as follows: In the firstpart, to study if ARV-induced apoptosis, viral replication, and the cleavage of σA intoσAC and σAN fragments are related to UPS, therefore, we will first test the effects onvirus internalization, viral replication, and cellular translation by UPS. Also, whetherUPS play a important role to regulate ARV-induced apoptosis, the expression ofssRNA- and dsRNA binding proteins (σNS and σA), and σA cleavage, thesequestions will be addressed in this study. In the second part, to explore themechanism of σA cleavage and translocation of σA into nucleus as well as thepossible function of the cleaved products, mapping of nuclear localization signal(NLS) and export localization signal (ELS) as well as understanding of the pathwayof translocation of σA into nucleus will be done in this study.

家禽里奧病毒 (avian reovirus, ARV)感染家禽造成多種疾病,包括病毒性關節炎、營養吸收不良症及慢性呼吸道疾病等,造成業者經濟損失。筆者研究團隊過去已證實ARV σC 蛋白具誘發細胞凋亡之功能。最近更進一步證實發ARV 在感染細胞後可經由活化Src kinase,再經由p53-mitochondrial pathway 引發下游一連串caspases 活化來誘發細胞凋亡。因此本研究將進ㄧ步探討ARV 感染宿主細胞後,是否利用ubiquitin-proteasome system(UPS)來調控其它機制以增加其本身利益。在我们前期試驗結果證實抑制細胞之UPS,可減少家禽里奧病毒所誘發之細胞病變、降低病毒力價及抑制病毒誘發細胞凋亡。這些新發現,使筆者研究團隊推測家禽里奧病毒在感染細胞後,UPS 可能扮演重要之調控角色,調控病毒誘發細胞凋亡、訊息傳遞、轉錄、蛋白質切割及病毒複製等。最近筆者研究團隊透過免疫螢光染色分析偵測到位於細胞核之家禽里奧病毒σA 蛋白,顯示此結構蛋白除了已知的功能外,仍可能具有其它生物功能。我们以RNA 干擾技術 (RNAinterference) 抑制家禽里奧病毒S-class 基因,結果發現一個未知產物的量隨著σA蛋白被RNA 干擾抑制而減少,但抑制病毒其它蛋白σC 或σNS,此未知產物的量則未受影響。初步證實部份σA 蛋白在胞內會被切割成兩個片段。由於過去研究顯示σA 蛋白無醣基化的轉譯後修飾及不會進入內質網,同時筆者研究團隊也證實σA 蛋白被切割的位置距離N 或C 端均有相當距離,因此排除σA 蛋白具有信號序列並在進入胞器後而被移除的可能性。發現σA 蛋白被切割成兩個片段(σAC及σAN)及進入核之現象,目前為止尚無任和相關研究報告發表。因此本研究另一個重點也將探討家禽里奧病毒σA 蛋白之切割是否與凋亡酵素、細胞內蛋白質切割酵素及UPS 調控有關,進一步探討σA 蛋白切割之機制、生物功能與對細胞及病毒本身之影響。綜合上述,本研究將進行如后兩大重點研究:1. 探討UPS 調控家禽里奧病毒複製與誘發細胞凋亡之機序及與σA 蛋白切割之關聯性: 首先將探討 UPS 對家禽里奧病毒進入細胞與病毒基因之轉錄及轉譯之影響。分析病毒attachment 及誘發細胞凋亡之σC 蛋白及與病毒複製有關之雙股及單股RNA 結合蛋白(σA 及σNS) 的表現情形及UPS 與σA 切割之關係。 探討ARV 究竟如何利用UPS?為何需要UPS?何時利用UPS?這些機序尚不清楚,值得深入探究。2. 探討σA 的切割及進入細胞核的機制及切割產物在細胞質與細胞核可能的功能: 首先定位σA 的切割位及nuclear localization signal(NLS)的位置與σA 進入細胞核的途徑及機制,再進一步研究σA 及其切割產物在細胞質與細胞核可能的功能。
其他識別: NSC97-2313-B005-048-MY3
Appears in Collections:分子生物學研究所

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