探討B型肝炎病毒核殼蛋白Serine 170位點磷酸化修飾之激酶及其對病毒複製之影響

Abstract

構成B型肝炎病毒(Hepatitis B virus, HBV)核殼(nucleocapsid)的核殼蛋白 (HBV core protein, HBc)為一磷酸化蛋白質，其C端的S155、S162、S170為三個最主要的磷酸化位點。近年研究顯示核殼蛋白的磷酸化狀態在HBV生活史中呈現動態變化，亦即核殼蛋白在進行pgRNA encapsidation前呈現高度磷酸化，但在進行pgRNA encapsidation以及後續包膜(envelopement)及分泌(secretion)至細胞外的複製過程中皆呈現去磷酸化狀態，意味著核殼蛋白的磷酸化與去磷酸化修飾在HBV生活史中扮演重要功能。然而，負責調控核殼蛋白磷酸化修飾的激酶(kinase)，和激酶所調控的特定磷酸化位點之功能仍不清楚。 本研究著眼於S170位點，首先利用基因體點突變的方式，將S170改變為Alanine及Aspartate，發現S170位點的磷酸化決定核殼蛋白是否能包裹pgRNA和合成DNA。接著利用回補的方式分別探討HBc及由SP1 splicing RNA所轉錄缺少C端最後一個胺基酸Cysteine之HBc-Cys蛋白中S170的功能，發現HBc及HBc-Cys蛋白對於DNA合成以及核殼組裝有不同的影響。為了驗證基因體點突變研究策略的結果，在不改變核殼蛋白胺基酸序列的情況下探討S170位點磷酸化的功能，我們嘗試尋找負責S170磷酸化之激酶及抑制劑。藉助於本實驗室所製備可專一性辨識去磷酸化S170之抗體，進行kinase inhibitor library之篩選，發現候選激酶Akt抑制劑的處理，會降低核殼蛋白的170位點磷酸化程度，進一步發現在Akt抑制劑處理下，HBV replicon所表現的disulfide bond-linkage HBc dimers和capsids及capsid所包裹的DNA皆顯著減少。以上結果表示Akt很可能是調控核殼蛋白磷酸化之激酶，目前正利用in vitro kinase assay來驗證此可能性，並進行確認特定Akt isoform，包括Akt1、Akt2及Akt3對HBc及HBc-Cys蛋白之磷酸化及其對調控HBV之複製過程，包括dimer/capsid之形成及pgRNA encapsidation之具體作用。

HBV core protein (HBc), the main structural protein of HBV icosahedral nucleocapsid, is a phosphoprotein. It contains three major conserved serine phosphorylation sites at S155, S162, and S170 in C-terminal domain. Recently, it was demonstrated that HBc undergoes dynamic phosphorylation during viral replication. HBc is highly phosphorylated before RNA encapsidation, which is dephosphorylated afterwards until envelopment and virion secretion. Therefore, HBc phosphorylation and dephosphorylation might play important role in viral replication. However, the putative kinase for specific Ser residue and the exact function still remains unclear. To focus on S170, we first took the genetic approach to generate the HBV replicons with S170A and S170D mutations. S170A but not S170D is unable to synthesize the capsid associated DNA, supporting the critical role of S170 phosphorylation in viral replicaion. Furthermore, we took complementation approach to analyze the individual function of S170 phosphorylation in HBc and SP1 splicing RNA encoded HBc-Cys, the two components in viral capsids, in viral replication. The results showed different function HBc and HBc-Cys in capsid assembly and DNA synthesis. Next, we have tried to identify the putative kinase and also the specific kinase inhibitor for S170, which might help study the function of S170 phosphorylation in the wild type HBc and HBc-Cys containing viral replicons. Aided by CS-170 Ab, which specifically recognizes the dephospho-S170 HBc, we screened a kinase inhibitor library and found that S170 the phosphorylation was decreased by a putative Akt inhibitor. The Akt inhibitor treatment decreased the expression of capsid protein, the capsid formation and also the capsid-associated DNA, suggesting the critical role of S170 phsophoayrlion in capsid assembly and DNA synthesis. The identity of Akt as the putative kinase for S170 is currently under investigation, approached by the in vitro kinase assay and si-RNA studuies. Moreover, the effect of specific isoform of Akt, including Akt1-3, on HBc and HBc-Cys in capsid assembly and DNA synthesis is also under invstigation. We expect the results can help clarify the kinase and the function of specific HBc-S170 phosphorylation in viral replication cycle.