探討調控嚴重急性呼吸道症候群病毒核殼蛋白磷酸化修飾之細胞訊息傳遞途徑

Abstract

SARS-coronavirus (SARS-CoV)為SARS (Severe acute respiratory syndrome) 的病源病毒，而nucleocapsid 則是SARS-CoV 的一種構造蛋白(structure protein)。過去關於冠狀病毒 nucleocapsid 的研究，指出此蛋白在冠狀病毒的生活史中扮演重要角色，可能具有多種功能並參與包括replication 及packaging 等過程的調控。 Nucleocapsid 是一個具有高度磷酸化修飾的蛋白，但造成磷酸化修飾的磷酸激酶（protein kinase）及其磷酸化修飾之影響卻始終未被清楚界定，只有Dr. Dales的研究團隊於 1996 年在 JHMV （mouse hepatitis virus, strain JHM ）的研究中曾經指出，PKA pathway活化也許與JHMV nucleocapsid 的磷酸化修飾有關，並可能影響病毒之 uncoating 過程。因此本論文以SARS-CoV 為研究對象，探討SARS-CoV nucleocapsid 在宿主細胞內發生磷酸化修飾 （phosphorylation） 的位置，以及可能造成磷酸化修飾的細胞內訊息傳導途徑 （cellular signaling pathway）。 本論文首先依據 Dr. Dales研究團隊的研究結果，試圖探討 PKA pathway 參與調控 SARS-CoV nucleocapsid磷酸化修飾之可能性。我們使用質體轉染，在 293T及 VeroE6兩種SARS-CoV可進行複製的細胞內表現SARS-CoV nucleocapsid，首先發現 SARS-CoV nucleocapsid 在這兩種細胞內主要屬於highly phosphorylated form。研究發現利用dbcAMP活化PKA pathway ，SARS-CoV nucleocapsid 的磷酸化修飾程度並沒有受到影響，但是我們發現當細胞發生 apoptosis時，活化的 caspase 可能造成 SARS-CoV nucleocapsid C-terminal 的 cleavage。 接下來我們利用質譜儀分析的方法訂定 SARS-CoV nucleocapsid 磷酸化修飾位置，同時使用磷酸激酶抑制劑（protein kinase inhibitors）篩檢的方法，系統性找尋可能參與 SARS-CoV nucleocapsid 磷酸化修飾的細胞內訊息傳導途徑。結果本論文尚未確定 SARS-CoV nucleocapsid 磷酸化修飾位置，但發現磷酸化修飾的位置極可能位於絲氨酸及精氨酸高度集中的區域。此外，使用磷酸激酶抑制劑篩檢方法的結果指出GSK3β pathway可能參與SARS-CoV nucleocapsid 的磷酸化修飾。同時，在質譜儀分析的過程中我們也發現幾個細胞內可能與 SARS-CoV nucleocapsid 結合之 protein，其可能之功能及受SARS-CoV nucleocapsid 磷酸化之影響值得進一步探討。 目前還需要進一步實驗才能確認細胞內的 GSK3β pathway 是否參與SARS-CoV nucleocapsid 的磷酸化修飾，但是可以預期的是，目前的研究結果將有助於了解 SARS-CoV nucleocapsid 磷酸化修飾在病毒生活史中的重要性，並以此為基礎，研究可能的抗病毒方法。

SARS coronavirus (SARS-CoV), a novel member of coronavirus, has been identified as the etiologic pathogen of the severe acute respiratory syndrome (SARS). The nucleocapsid (N) protein of coronavirus, one of its structure protein, was proposed to play a critical role in the virus life cycle through affecting a variety of processes, including the replication and packaging etc. Nucleocapsid is well known as a highly phosphorylated protein. However, both the putative kinase(s) attributed to its phosphorylation and the effect of the phosphoryaltion on viral life cycle remained unidentified. Only the group led by Dr. Dales has reported that in JHMV（mouse hepatitis virus, strain JHM ） the PKA pathway might involve in the N phosphorylation and affect the virus uncoating process. Therefore, in the current study, we proposed to use the SARS-CoV as the assay system to study the phosphorylation sites of its N protein and also the cellular signaling pathway(s) affecting its N phosphorylation. Based on the information derived from Dr. Dales group, we first tried to investigate the plausible role of PKA signaling on the N phosphoryaltion of SARS-CoV. By transfecting the N expression plasmid construct into 293T and VeroE6 cell lines, we found that N protein is highly phsophorylated in both cell lines. We next treated the cells with dbcAMP to activate the PKA pathway and evaluated the effect on the N phosphorylation status. The result did not support the involvement of PKA pathway in regulating the SARS-CoV N phosphorylation. However, we have identified a cleavage of N protein at the C terminal, which was induced by the apoptosis process. In the second part of the thesis, we tried to determine the phsophorylation sites of SARS-CoV N by mass spectrometry analysis and also tried to determine the signaling pathway(s) involved in its phosphorylation by systemically screening with the inhibitors for specific protein kinases. Although the phosphorylation sites have not yet been conclusively determined, the results suggested that the major phosphoryation sites might locate at the central serine-arginine rich region. Furthermore, the screening results pointed out that GSK-3β signaling pathway might involve in the phosphorylation of SARS-CoV N protein. In this process, we also have identified several cellular proteins associated with N protein. Their functional effect on virus life cycle and moreover the effect of N phosphoryaltion on their association is worthy to be further investigated. The current results pointed out a plausible cellular signaling pathway on the phosphoryaltion of SARS-CoV N. The generality of the effect of GSK-3β signaling on regulating the phosphorylation of other coronviruses awaits further investigation. The involvement of GSK-3β signaling in the phosphoryaltion of SARS-CoV N also needs to be verified by some other assays instead of inhibitor only. Prospectively, the results might help investigate the effect of N phosphorylation on viral life cycle and moreover point out another target for designing of anti-viral strategy.