抑制第二型鈣調蛋白依賴激酶δ降低非結構蛋白5A的高度磷酸化以及C型肝炎病毒複製

Inhibition of CaMKII δ Reduced NS5A Hyperphosphorylation and HCV Replication

陳逸鴻(Yi-Hung Chen)

指導教授 ： 余明俊

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10971

摘要

根據實驗室先前的研究，我們鑑定出一個位於C型肝炎病毒(Hepatitis C virus, HCV)非結構性蛋白5A (Nonstructural protein 5A, NS5A)上的全新磷酸化位點(S1位點)，並且發現此位點的磷酸化為病毒RNA複製所必需。此觀察結果促使我們進一步找尋負責此位點磷酸化之磷酸化激酶，因為藉由抑制磷酸化激酶可為治療C型肝炎提供另一替代療法。從電腦演算法預測結果發現鈣調蛋白依賴激酶可能對S1位點進行磷酸化。當我們抑制病毒感染之肝細胞株中的鈣調蛋白(Calmodulin)，S1位點之磷酸化程度與病毒RNA數量皆受到抑制。對肝細胞株處理鈣調蛋白依賴激酶II (calmodulin dependent protein kinase II, CaMKII)抑制劑亦獲得相似的結果。藉由反轉錄(Reverse transcription)與聚合連鎖反應(PCR)進一步確認肝細胞株會表現CaMKII γ和δ兩種異構體(Isoform)。抑制肝細胞株內的CaMKII δ表現會進而抑制病毒RNA的數量，而抑制CaMKII γ的表現則沒有此現象，因此我們認為CaMKII δ可能參與S1位點的磷酸化以及抑制CaMKII可以進一步降低S1位點之磷酸化和病毒RNA的數量。

關鍵字

鈣調蛋白 ； 鈣調蛋白依賴激? ； HCV RNA 複製機轉 ； 宿主激? ； HCV療法

並列摘要

We previously identified a novel phosphorylation site S1 in the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) required for HCV replication. These observations provide us the impetus to look for host kinases responsible for its phosphorylation because inhibition of the kinases may be an alternative method for HCV treatment. By using computer algorithms, calmodulin dependent protein kinase (CaMK) was predicted to phosphorylate the S1 site. The S1 site phosphorylation levels and HCV RNA levels were decreased in a dose-dependent manner when we treated infected cells with calmodulin inhibitor (W7). Inhibition of the calmodulin dependent protein kinase II with the inhibitor KN93 produced similar results as the W7 treatment. Reverse transcription and PCR results indicated expression of CaMKII δ and CaMKII γ in the Huh7.5.1 cells. CaMKII δ knockdown, not CaMKII γ knockdown, decreased the HCV RNA in the HCV-infected cells. We conclude that CaMKII δ may be a host kinase responsible for NS5A S1 site phosphorylation and that inhibition of CaMKII diminishes NS5A S1 phosphorylation levels as well as HCV RNA levels in HCV-infected Huh7.5.1 cells.

並列關鍵字

Calmodulin ； Calmodulin dependent protein kinase II ； HCV replication ； Host kinase ； HCV therapy

參考文獻

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