DDX3 移位至次細胞巨分子結構的功能性角色及其對C型肝炎病毒感染之影響

Abstract

DEAD box RNA 解螺旋酶DDX3，亦稱為CAP-Rf，為一可與C 型肝炎核心蛋白 發生交互作用的細胞因子。多年來，本實驗室致力於闡明該解螺旋酶的生物功能。目前 本實驗室的研究成果顯示：DDX3 具多重生物功能，除可作為轉錄活化因子、抑制 cap-dependent 轉譯起始作用外，亦為一可調控細胞之生長之抑癌基因。近年來，亦有 多篇文獻報導DDX3 參與mRNA 運送及RNA 剪接的進行。然而，DDX3 如何調控及 執行其多樣化生物功能的分子機制尚待研究。近來，我們初步的觀察顯示DDX3 可出現 於兩種次細胞巨分子結構stress granules (SGs) 與PML nuclear bodies (PML-NBs)中。此 外，我們亦發現DDX3 可與此二結構之標記蛋白PABP1 與PML 有交互作用存在。SGs 為細胞遭受環境壓力時，出現於細胞質內顆粒狀之RNA 結構，其由48S preinitiation complexes、mRNAs 及RNA 結合蛋白所構成。一般認為在細胞遭受環境壓力時，其可 調控細胞中mRNA 之代謝。PML-NBs 由超過40 種細胞因子與PML 所組成。與其他細 胞核中特化之次細胞結構不同的是，PML-NBs 的功能並不專一且參與多種細胞功能， 諸如：轉譯調控、細胞老化與凋亡。由於DDX3 為一轉錄與轉譯之調控因子，因此這些 結果暗示DDX3 可能參與SGs 與PML-NBs 之生物功能，且DDX3 在細胞中不同的分 布位置可能調控其本身多樣化之生化功能。為此，本三年研究計劃將探討DDX3 位於此 兩種次細胞巨分子結構SGs 與PML-NBs 之功能角色。本計劃之研究方向分述如下。 為確認DDX3 於SGs 與PML-NBs 之分布，我們將進一步測試DDX3 與SGs 及 PML-NBs 標記蛋白的交互作用與分布位置。我們並將定位出這些交互作用與移動至 SGs 及PML-NBs 所需之DDX3 蛋白片段。此外我們亦將利用顯微鏡擷取影像以探討 含有DDX3 之SGs 及PML-NBs 的動態形成與分解。由於DDX3 參與轉錄與轉譯過 程，因此DDX3 是否參與SGs 及PML-NBs 相關之轉譯與轉錄作用亦為我們研究之重 點。此外，有鑑於後轉譯修飾作用亦可影響受修飾蛋白與其他蛋白交互作用的發生以及 該蛋白之核酸結合能力，因此我們將採用蛋白質體學方法以尋找DDX3 蛋白上之後轉譯 修飾的種類、位置及其功能角色。 雖然DDX3已被證實為C型肝炎病毒複製所必須且與C型肝炎病毒核心蛋白具有 交互作用，然而其作用機制至今仍未明瞭。由於SGs 及PML-NBs為宿主細胞對抗病毒 感染反應的一環，且為多種病毒感染過程中攻擊的目標，是故C型肝炎病毒可能藉由與 這些含有DDX3之次細胞結構的交互作用破壞宿主細胞的防護機制。因此，本研究計畫 的另一目標在於探討C型肝炎病毒感染過程中DDX3移位至次細胞結構之功能角色。我 們將探討C型肝炎病毒感染對DDX3、SGs 及PML-NBs於細胞中分布之影響。我們亦將 闡明C型肝炎病毒蛋白、DDX3、SGs 與PML-NBs的功能性交互作用並進一步探討其 作用機制。 綜合上述，本研究計畫試圖進一步闡明DDX3 多樣化生物功能的調控機制，將 有助於進一步釐清C 型肝炎病毒的致病機轉，並提供C 型肝炎病毒研究之基礎資訊， 以利於病毒感染及肝腫瘤之治療策略的研發。

The DEAD-box RNA helicase DDX3 was originally isolated as hepatitis C virus (HCV) core-interacting protein by several laboratories including ours. Over the past years, my lab has investigated the biological function of DDX3. Our recent findings indicated that DDX3 possesses pleiotropic functions, such as acting as a transcriptional activator, a negative regulatory role in the cap-dependent translational initiation, and a cell growth regulator involved in tumorgenesis. Additionally, a growing number of reports have shown that DDX3 can exert a multiplicity of effects on cellular processes including mRNA export and RNA splicing. However, the molecular mechanism as to how DDX3 achieves these plethoric functions is not well understood. Recently, our preliminary data revealed that DDX3 was sequestered in two specialized subcellular macromolecular structures, stress granules (SGs) and PML nuclear bodies (PML-NBs). Moreover, we also found DDX3 interacts with SGs and PML-NBs markers, PABP1 and PML, respectively. SGs are cytoplasmic granular RNA structures in response to environmental stress. SGs, containing stalled 48S preinitiation complexes, heterogenous mRNAs and other RNA-binding proteins, are postulated to play a critical role in regulating mRNA metabolism during stress. The PML-NB is composed of more than 40 cellular factors and the essential component PML (promyelocytic leukaemia) tumour suppressor protein. Unlike other more specialized subnuclear structures, PML-NBs are functionally promiscuous and have been implicated in the regulation of diverse cellular functions, such as transcriptional regulation, cell senescence and apoptosis. Given that DDX3 acts as a transcriptional and translational regulator, these observations suggest that DDX3 is engaged in SGs- or PML-NBs-associated processes. Therefore, it is likely that the translocation of DDX3 may play a role in modulation of its diverse biological function. Thus, knowledge of the biological functions of these two DDX3-containing subcellular macromolecular structures may highlight the regulatory mechanisms of DDX3 pleiotropic functions. In view of these, the objective of this project in the next three years will decipher the functional roles of DDX3 located in two subcellular macromolecular structures, SGs and PML-NBs. The specific aims which we intend to accomplish are stated below. In the aspect for confirming whether DDX3 could be recruited into SGs and PML-NBs, we will first further examine the colocalization and interactions among DDX3 and the SGs and PML-NBs marker proteins. If the result supports this notion, further mapping of the sequence elements within DDX3 required for these interactions and sequestering into SGs and PML-NBs will be carried out. Moreover, examination of the dynamics of DDX3-containing SGs and PML-NBs as monitored by time-lapse microscopy will be pursued. Since our previous study indicated that DDX3 is involved in transcription and translation processes, whether these biological functions of DDX3 are involved in the SGs- and PML-NBs-associated processes will also be explored. Additionally, in viewing that post-translational modifications (PTM) can modulate the ability of proteins to interact with their partners or alter the DNA/RNA binding affinity of transcription factor, a global search of the PTM profile of DDX3 will also be carried out. Although the action mechanism is still obscure, DDX3 is reported to be required for HCV replication and interacts with HCV core protein. Considering that both SGs and PML-NBs are involved in host antiviral response and targeted by various viruses, it seems very likely that HCV evolves to impede host protective measures through the association of DDX3-containing subcellular complex. Therefore, another line of this project will focus on the functional role of DDX3 translocation to subcellular macromolecular structures in HCV infection. Briefly, this part will include the delineation of the effect of HCV infection on the cellular distribution of DDX3, SGs and PML-NBs. Efforts will specifically direct to the functional interactions among HCV viral proteins, DDX3, SGs and PML-NBs. The results of this study will provide the mechanistic explanation for the effect of DDX3 on HCV replication. Collectively, the approaches outlined above will allow envisaging how the pleiotropic functions of DDX3 are regulated, which may be informative for the future research on HCV replication and ultimately development of preventive or therapeutic strategies for HCV infection and hepatocellular carcinoma.