C 型肝炎病毒非結構蛋白質 5A 高度磷酸化需要非結構 蛋白質 3 解旋酶三磷酸腺苷結合之活性

Abstract

Ｃ型肝炎病毒(Hepatitis C virus)的非結構蛋白5A(non-structural protein 5A, NS5A)是一個磷酸化蛋白，它有兩個磷酸化態：低度及高度磷酸化態。兩者都對HCV生命週期至關重要。 NS5A通過高度保守的絲氨酸位點（即S225，S229，S232和S235）以連續磷酸化方式從低磷酸化狀態轉變為高磷酸化狀態。然而S225的磷酸化是由哪個蛋白質磷酸化仍未知。許多文獻指出NS5A的高度磷酸化取決於NS3(non-structural protein 3, NS3)，NS3為具有蛋白酶（protease）及解旋酶（helicase）雙重功能的蛋白質。首先，必須當NS3, NS4A, NS4B及NS5A轉譯在同一條多蛋白質上才會發生NS5A的高度磷酸化。再來，當NS3自行切割後NS5A才會高度磷酸化。最後，NS5A的高度磷酸化可以發生在沒有激酶的體外試驗中。這使我們推測是否當NS3蛋白酶在切割多蛋白質中的NS5A時，會使結合三磷酸腺苷的NS3解旋酶將其水解的磷酸根轉移至NS5A。在本篇的研究探討NS3解旋酶是否可以作為激酶磷酸化NS5A。我們利用突變的方式扼殺了NS3解旋酶三磷酸腺苷結合及水解的能力，結果顯示NS5A的高度磷酸化及NS5A上S225、S229、S232、S235和S238的磷酸化程度大幅降低。另外，NS5A的高度磷酸化及這些位點的磷酸化無法用正常功能的NS3解旋酶來補救。綜合上述結果，顯示NS3解旋酶對於NS5A的高度磷酸化是必要的。接著利用激酶不敏感性的N6-芐基-三磷酸腺苷-伽馬-硫(N6-benzyl-ATP-g-S)的三磷酸腺苷類似物作為三磷酸腺苷的材料，再透過特異性抗硫代磷酸化抗體進行磷酸根的追蹤，我們檢測到 NS5A 上的硫代磷酸化為高度磷 酸化。當 NS3 上的關鍵三磷腺苷結合位點發生突變時，硫代磷酸化顯著降低。這 些結果顯示 NS3 解旋酶參與在 NS5A S225/S229/S232/S235 的磷酸化的鏈鎖反應而 造成 NS5A 的高度磷酸化。總之，我們的結果提供了關於病毒如何在其生命週期中 調節蛋白質磷酸化以及解旋酶功能的新觀點。

The non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with hypo- or hyper-phosphorylation states. Both are critical to the HCV life cycle. NS5A transits from hypo- to hyper-phosphorylated state via phosphorylation of a cluster of highly conserved serine sites i.e. S225, S229, S232, and S235 in a cascade manner. How the initial phosphorylation occurs on S225 remains unknown. Several lines of evidence indicate that NS5A hyper-phosphorylation could be mediated by NS3, another non-structural protein that has both protease and helicase activities. First, hyper-phosphorylation only occurs when NS5A is encoded on a polyprotein alongside with NS3 and other viral non-structural proteins (NS4A and NS4B). Second, NS5A hyper-phosphorylation requires NS3-mediated intra-molecular auto-cleavage at the NS3-NS4A junction. Third, in vitro transcription/translation assay result suggests that NS5A hyper-phosphorylation occurs in the absence of host kinases. All these evidences prompted us to test whether the ATP-binding activity of the NS3 helicase may be involved in NS5A hyper-phosphorylation. By mutating key helicase sites involved in ATP-binding and ATP-hydrolysis, we found that NS5A hyper-phosphorylation and phosphorylation at serine sites S225, S229, S232, S235, and S238 were significantly reduced. Co-transfecting the helicase-defective NS3 helicase with a wild-type NS3 did not restore NS5A hyper-phosphorylation in trans. Using a kinase-insensitive N6-benzyl-ATP-g-S analog that allowed phosphate tracking by a specific anti-thiophosphorylation antibody, we detected thiophosphorylation on NS5A that corresponded to hyper-phosphorylation. The thiophosphorylation was significantly reduced when the key ATP-binding site on NS3 was mutated. We conclude that the NS3 helicase is involved in NS5A S225/S229/S232/S235 phosphorylation cascade that leads to NS5A hyper- phosphorylation. Our results provide new perspectives on how viruses regulate protein phosphorylation during their life cycle and on the yet fully unraveled functions of helicase.