冠狀病毒核殼蛋白磷酸化修飾調控病毒 RNA 轉錄過程之分子機制研究

The Mechanism for the Phosphorylation of Coronavirus Nucleocapsid Protein in Regulating the Discontinuous RNA Transcription Process

吳佳欣(Chia-Hsin Wu)

指導教授 ： 葉秀慧

國立臺灣大學/醫學院/微生物學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.01104

摘要

冠狀病毒為具有套膜的 RNA 病毒，含有一全長約∼30 kb的正股單鏈 RNA 基因 (gRNA)，可轉譯出多個參與病毒複製的非結構蛋白。除了全長 RNA 基因外，冠狀病毒的複製過程中，亦會產生數個片段較短、可轉譯出結構蛋白的亞基因 RNA (sgmRNA)。所有的病毒RNA，包括gRNA和sgmRNAs，在5’ / 3’兩端序列皆相同，乃是透過一種在合成負股 RNA 時獨特的不連續轉錄機制而產生的。此一過程是由轉錄調節序列 (TRS) 所控制，TRS的位置則分別座落於前導序列 (稱為 leader TRS) ，和在每一個基因前面（稱為 body TRS）。目前認為 sgmRNAs是在負股 RNA 合成過程，藉由leader TRS和互補股body TRS之間的鹼基配對，造成模板切換而發生。然而，調控冠狀病毒進行不連續轉錄到連續轉錄的分子機制仍然不甚清楚。
病毒生活史中表現量最高的sgmRNA可轉譯出核殼蛋白，核殼蛋白被分類為結構蛋白，可與病毒基因纏繞且螺旋包裹入病毒顆粒內。此外，透過反向遺傳學研究，發現核殼蛋白也可調控病毒 gRNA 的合成，但其機制仍然不清楚。已知核殼蛋白是具磷酸化修飾的高鹼性蛋白，而我們的研究發現，在兩種冠狀病毒：SARS冠狀病毒和JHMV鼠冠狀病毒，核殼蛋白的磷酸化修飾主要發生於絲氨酸-精氨酸高度集中區域的絲氨酸位點上。我們同時確認了 GSK-3 為主要負責此磷酸化修飾的磷酸激酶。利用 GSK-3 抑製劑可有效降低感染後病毒效價和感染所造成的細胞病變效應，暗示核殼蛋白的磷酸化與病毒複製過程相關。
本篇研究中更發現經 GSK-3 介導產生磷酸化修飾的核殼蛋白參與 JHMV 不連續轉錄與連續轉錄過程間的轉換。抑制 GSK-3 介導產生磷酸化修飾的核殼蛋白會使得 gRNA 和較大 sgmRNAs 的表現量減少；但較小的 sgmRNAs 合成不受影響。因此，具磷酸化修飾的核殼蛋白可能參與確保全長gRNA產生以及成功生產的成熟病毒顆粒的過程。我們發現細胞內的 DDX1 會被吸引到具磷酸化修飾核殼蛋白的蛋白聚合體以利於 RNA 連續轉錄過程。我們的研究成果證明，冠狀病毒可藉由 GSK-3 介導產生磷酸化修飾的核殼蛋白來參與不連續轉錄過程的調控。

關鍵字

冠狀病毒 ； 核殼蛋白 ； 磷酸化修飾 ； 不連續轉錄 ； 嚴重急性呼吸系統綜合症

並列摘要

Coronaviruses (CoVs) are enveloped RNA viruses containing a ~30 kb positive-sense single-stranded genomic RNA (gRNA), which encodes non-structural proteins involved in viral replication. In addition to the gRNA, several subgenomic mRNAs (sgmRNAs) are generated during viral replication, which encode mainly the structural proteins. All the viral RNAs, including the gRNA and the sgmRNAs, are co-terminal, through a unique discontinuous transcription mechanism during negative-strand RNA synthesis. This discontinuous process is controlled by a conserved transcription regulating sequence (TRS), which is located after the leader sequence (leader TRS) and in front of each gene (body TRS). It has been suggested that through base pairing between the leader TRS and the complementary body TRS, a template-switching event occurs to generate the discontinuous sgmRNAs. However, the molecular mechanisms controlling the switch from discontinuous to continuous transcription in CoV still remain unknown currently.
The most abundant viral sgmRNA encodes the viral nucleocapsid (N) protein, which is categorized as a structural protein to form a helical ribonucleoprotein required for packaging gRNA into the virion. N protein also has non-structural functions in regulating the synthesis of viral gRNA as revealed by reverse genetic studies, but the underlying mechanism is remained unclear. Regarding to the N protein as a highly basic protein with substantial phosphorylation modifications, our study demonstrated that the major phosphorylation sites in both SARS-CoV and JHMV are the Ser residues clustered within the central serine–arginine (SR)-rich motif. We also identified the GSK-3 to be the kinase responsible for this phosphorylation. Treatment with GSK-3 inhibitor can reduce the viral titer and cytopathic effects, suggesting this phosphorylation in N protein is relevant to the viral replication cycle.
In the current study, we further discovered a novel function of this GSK-3 mediated N phosphorylation in supporting the transition from discontinuous to continuous transcription of JHMV. Suppression of this specific phosphorylation diminished the synthesis of gRNA and larger sgmRNAs but not the smaller ones. It thus suggested this N phosphorylation might participate in the discontinuous transcription process, with function to ensure the synthesis of full length gRNA and successful production of mature virions. We found the cellular DDX1 is recruited to the phosphorylated N-containing complex and facilitates template readthrough when encountering TRSs for the synthesis of longer viral RNAs. Our results thus demonstrate a unique strategy for the transition from discontinuous to continuous transcription in CoVs via the novel function of the GSK-3 phosphorylated viral N protein.

並列關鍵字

coronavirus ； SARS ； nucleocapsid ； phosphorylation ； discontinuous transcription

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