臺灣博碩士論文加值系統

C型肝炎病毒會引發慢性肝炎並進一步的造成肝硬化及肝癌。許多病毒，像是B型肝炎及C型肝炎病毒皆會利用宿主的PI3K-Akt的細胞訊息傳遞途徑來抑制宿主細胞自身的凋亡。Akt也就是所謂的Protein kinase B是PI3K下游的一個激脢，其下游調控了許多細胞內重要的訊息傳遞，因而他的活化與否便可以調控細胞的生長，增生，代謝及細胞凋亡。而這麼樣一個可以磷酸化絲氨酸或是酥氨酸的激脢在細胞中的影響可謂舉足輕重。C型肝炎病毒的非結構性蛋白5B 是病毒的RNA聚合脢，用以複製病毒的RNA。以昆蟲細胞表現的NS5B 可以被 Protein Kinase C related kinase 2磷酸化其上的Serine。然而卻沒有相關的研究指出NS5B在肝臟細胞中是否會被磷酸化。而藉由比對已知的C型肝炎資料庫我們發現在NS5B上具有一個Akt可磷酸化共通序列RXRXXS/T且此Akt基質共通序列位在NS5B的C端酵素作用區中。而此序列中Akt可能會磷酸化的絲氨酸位置則是位在NS5B上第506個胺基酸的位置 (S506)。然而此Akt基質共通序列僅出現在基因型1的C型肝炎病毒NS5B上。因此我們將基因型1(S506)及不含有此序列的基因型2 (A506)分別置入可以以哺乳類細胞表達的載體中，另外，利用點突變將此基因型上Akt可能的磷酸化點進行突變 (S506A，S506D)來進行比較。
利用免疫沉澱法，我們證明了基因型1的C型肝炎病毒NS5B不論是在肝臟細胞Huh7或是非肝臟細胞的人類胚胎腎臟細胞 (293T)中都會和Akt產生交互作用。而突變的NS5B-1b (S506A)及基因型2的NS5B也會和Akt結合但是其結合的能力較wild type的NS5B為弱。利用共軛焦顯微技術來觀察細胞內此兩種蛋白的交互作用，也可以發現其兩者會在細胞內共定位。且利用大腸桿菌表現出來的GST-NS5B蛋白來和Akt進行試管反應也證實了Akt和NS5B的確具有交互作用。以NS5B deletion mutant找出了Akt在NS5B上的結合位置在姆區 (Thumb domain)也就是(372-591)的位置，且這段作用區中有我們預測的Akt磷酸化位置。更重要的是不論NS5B全長或是姆區皆能和活化態的Akt結合，暗示著NS5B的確可能是Akt的受質。研究有關NS5B與Akt作用所帶來的影響仍在持續進行，希望能夠解開Akt和NS5B的作用在生物學上是否帶來什麼樣的影響，如C型肝炎病毒的複製或是宿主細胞內的訊息傳遞是否會改變，以帶來更多有關於肝炎療法的進步。

Hepatitis C virus (HCV) is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The phosphatidylinositol 3-kinase (PI3K)–Akt pathway is utilized by many viruses, including HBV and HCV, for inhibition of apoptosis of infected cells. Akt, a serine/threonine kinase, is a substrate of PI3K and acts as an important signal mediator of cell survival following activation. The HCV NS5B, a putative serine phosphoprotein, is the viral RNA-dependent RNA polymerase (RdRP) required for replication of the HCV RNA genome. The phosphorylated form of HCV NS5B was observed when expressed in insect cells using a recombinant baculovirus. Though protein kinase C-related kinase 2 was implicated to be responsible for HCV NS5B phosphorylation, phosphorylation of HCV NS5B in human liver cells has never been demonstrated. Previously, we noticed that there is a putative Akt-phosphorylation motif, (RXRXXS/T), containing the potential phosphorylated serine residue at amino acid 506 and two arginine residues at amino acid 501 and 503 in the catalytic core region of HCV NS5B. Most importantly, this motif is highly conserved in HCV genotype 1 but not in non-1 genotype. Therefore, we constructed several mammalian vectors expressing NS5B, including wild type, S506A, S506D, from HCV genotype 1b and genotype 2b (A506). By using co-immunoprecipitation, we demonstrated that NS5B of HCV genotype 1b interacts with Akt in both non-hepatocytes-derived 293T and hepatocyte-derived Huh7 cell lines. NS5B-1b mutant (S506A) and NS5B-2b also bound Akt but with weaker affinity than NS5B-1b (S506). The in vivo interaction of NS5B-Akt was further verified in confocal microscopy study. By using GST pull down assay, the in vitro interaction of NS5B and Akt was also demonstrated. The Akt binding site on NS5B could be mapped to the thumb domain of NS5B (372-591), the region containing the putative Akt phosphorylation motif. Most importantly, both full length (1-591) and the thumb domain of NS5B (372-591) bind the activated Akt with its serine phosphorylated form, implicating that NS5B might be a substrate of Akt. Further study aiming to elucidate the biological significance of the interplay between NS5B and Akt and its effect on HCV replication and host signaling pathways is ongoing. Hopefully, understanding of the interplay of HCV NS5B and Akt may also facilitate new therapeutic strategy development for HCV infection.

Table of Content
Chinese Abstract 1-2
English Abstract 3-4
Table of Content 5
Introduction 6-10
Material 11-16
Methods 17-21
Result 22-24
Discussion 25-28
Figure 29-34
References 35-37

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