C型肝炎病毒非結構蛋白質3解旋酶具有蛋白激脢之功能

Abstract

生物體藉由蛋白質的磷酸化來調控相當多細胞中的訊息傳遞路徑。以丙型肝炎病毒（Hepatitis C virus）為例，非結構蛋白5A (non-structural protein 5A)的磷酸化就參與在病毒基因複製的調控路徑當中。我們團隊先前發現，非結構蛋白5A從低度磷酸化態轉換成高度磷酸化態，需要非結構蛋白5A上高度保留的絲氨酸位點（即S225、S232、S235和 S238）進行連續地序列性磷酸化。然而作為起始訊號的S225磷酸化是如何發生的目前仍未知。蛋白質激酶常被視為進行磷酸化蛋白質的必要酵素。但是在生物體中，已有相當多不需要蛋白質激酶就能完成蛋白質磷酸化的例子。例如：P型三磷酸腺苷酶（P-type ATPases）及雙分子磷酸化訊息傳遞系統（two-component phosphorylation relay system）。過去已有報導指出非結構蛋白5A的高度磷酸化，需要非結構蛋白3 (non-structural protein 3) 與其轉譯在同一條多蛋白質上才會發生，而我們先前的研究也發現除此之外，非結構蛋白3解旋酶具有三磷酸腺苷酶的結合能力也會影響非結構蛋白5A的高度磷酸化。在本篇的研究當中，我們發現如果突變負責非結構蛋白3結合三磷酸腺苷酶能力的位點(K210N、D290N、E291Q和H293A) 或是突變負責非結構蛋白3水解三磷酸腺苷酶的能力的位點(Q460A、R464A 和 R467)，都會大幅降低非結構蛋白5A上絲氨酸位點（S225、S232、S235和S238）的磷酸化，相反地，如果是突變在非結構蛋白3上負責結合核糖核酸的位點（W501A），則不會影響非結構蛋白5A的磷酸化。上述的實驗結果顯示：非結構蛋白3結合及水解三磷酸腺苷酶的能力對於這些絲氨酸的磷酸化（S225、S232、S235和S238）相當重要。接著我們在這些絲氨酸位點中（S225、S229、S232和S235）任取三個做丙氨酸突變（SAAA、 ASAA、AASA和AAAS），結果顯示只有S225可以在其他絲氨酸位點無法被磷酸化的情況下（SAAA），可以被磷酸化，上述的結果與S225的磷酸化是開啟後續序列磷酸化訊號的論點一致。體外激酶試驗顯示非結構蛋白3可以直接磷酸化S225，但無法磷酸化S229、S232和S235。只有加入第一型酪蛋白激酶(casein kinase I isoform alpha, CKI alpha)在含有非結構蛋白3的體外激酶試驗中才能看到S229、S232和S235的磷酸化。我們由上述實驗結果得知非結構蛋白3解旋酶可以磷酸化S225，並藉此開啟下游由第一型酪蛋白激酶催化的一連串序列性磷酸化。

Protein phosphorylation regulates many biological processes in organisms. In the case of the hepatitis C virus (HCV), phosphorylation of its non-structural protein 5A (NS5A) mediated viral genome replication. Our group previously showed that NS5A transits from the so-called hypo-phosphorylated to hyper-phosphorylated state via phosphorylation in a cluster of highly conserved serine sites S225, S232, S235 and S238 in a sequential manner. However, how the initiating phosphorylation event on S225 occurs remained unknown. Protein kinases have been the conventional enzymes responsible for protein phosphorylation; however, there are examples in biology where protein phosphorylation does not require a protein kinase such as the P-type ATPases and the so-called two-component phosphorylation relay system. It has been reported that NS5A hyper-phosphorylation requires NS3 encoded on the same NS3-NS5A poly-protein molecule. Our previously data showed that NS3 ATP-binding activity was required for NS5A hyper-phosphorylation. In the present study, I found that both mutations (K210N, D290N, E291Q, H293A) that disrupt ATP-binding and mutations (Q460A, R464A and R467) that disrupt ATP hydrolysis activity reduced NS5A hyper-phosphorylation at serine sites S225, S232, S235, and S238 in NS3-5B transfected T7-huh7 cells. In contrast, mutation in W501A that destroyed RNA-binding activity did not affect NS5A phosphorylation. The above results are consistent with a role of NS3 ATP-binding and hydrolysis activity in S225 phosphorylation that fires sequential S232/S235/S238 phosphorylation. Alanine mutation (SAAA, ASAA, AASA, AAAS) among any three of the four serine residues (S225, S229, S232, and S235) showed that only S225 could be phosphorylated when other serine residues could not be phosphorylated, in line with S225 phosphorylation as the initiation signal for sequential phosphorylation. In vitro kinase assay showed that purified NS3 directly phosphorylated S225 but not S229, S232, and S235. Phosphorylation at the other serine residues was detected only when casein kinase I alpha (CKI alpha) was included in the in vitro NS3 activity assay. We concluded that the NS3 helicase could phosphorylate NS5A in vitro at S225, which then primes sequential phosphorylation at S232/S235/S238 by CKI alpha.