核殼蛋白質在中東呼吸症候群冠狀病毒RNA包裹訊號辨識及病毒組裝中扮演的角色

Abstract

中東呼吸症候群冠狀病毒(MERS-CoV)是新型感染人類的冠狀病毒，患者感染後會引起類似感冒症狀外加嘔吐、腹瀉等腸胃道症狀，重症則出現急性腎衰竭、血管內瀰漫性凝血等併發症，致死率高達35%。過去研究已知包裹訊號 (package signal)對於冠狀病毒包裹其RNA基因體是非常重要的。先前我們實驗室利用基因體序列分析比對與RNA二級結構預測之方式預測了MERS-CoV的包裹訊號可能出現在核酸序列19,712-19,969範圍之中。利用類病毒顆粒(virus-like particle)的系統觀察，發現包裹訊號確實在這段258個核酸的序列之中，因此將此段RNA命名為PS258(19712-19969)ME，研究也發現包裹RNA需藉由核殼蛋白質(nucleocapsid protein; N protein)達成。本研究接續先前的研究，尋找PS258(19712-19969)ME中更精確的包裹訊號所在，探討核殼蛋白質與其結合的RNA-binding domain，也同時探討另一預測含有穩定二級結構，由核酸序列20,022-20,173長152個核酸組成的 RNA152(20022-20173)ME片段是否也可作為包裹訊號。同樣使用類病毒顆粒系統觀察，結果發現包含於PS258(19712-19969)ME中由核酸序列19,801-19,864組成的二級結構PS258ME-SLII與RNA152(20022-20173)ME這兩段序列皆有單獨作為包裹訊號並將RNA包裹到類病毒顆粒的能力。另一方面將帶有PS258ME-SLII頂端二級結構的SL19805ME片段與純化的MERS-CoV核殼蛋白質全長及其他sub-domains，利用filter-binding assay與electrophoretic mobility shift assay方式皆可以觀察到核殼蛋白質的N端和C端與SL19805ME RNA的結合。而利用IP的方式，初步看到核殼蛋白質與膜蛋白質(membrane protein; M protein)有交互作用，但是否藉此交互作用將帶有RNA基因體的核殼蛋白質包裹至病毒顆粒內則需進一步證實。關於MERS-CoV如何將帶有包裹訊號的RNA片段包裹至病毒顆粒內，以及結構蛋白質彼此之間參與在病毒組裝上的詳細機制則需要更多的實驗來進行探討。

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus. The symptoms of the infected patients include cold symptoms, vomiting, diarrhea and gastrointestinal symptoms. Severe cases may suffer from acute kidney failure and disseminated intravascular coagulation (DIC). The mortality rate is up to 35%. In previous studies, the importance of RNA package signal in coronavirus RNA genome packaging has been well-established. Based on genome sequence alignment and RNA secondary structure prediction, our laboratory has previously predicted a potential package signal of MERS-CoV located at nt 19,712 to 19,969. By using a MERS-CoV virus-like particle (VLP) system, the result showed that the 258-nt fragment, named PS258(19712-19969)ME, is sufficient to function as a package signal. In addition, the package is nucleocapsid (N) protein-dependent. In the present study, the minimum sequence and structure within the PS258(19712-19969)ME to function as a package signal was analyzed and the RNA-binding domain of N protein was determined. Meanwhile, another potential fragment, RNA152(20022-20173)ME, spanning nt 20,022-20,173 with stable secondary structure was examined for its function to act as a package signal. Results demonstrated that both PS258ME-SLII, a 64-nt substructure of PS258(19712-19969)ME, and RNA152(20022-20173)ME can independently act as a package signal and be packaged into VLPs. In addition, a RNA probe SL19805ME representing the top part of PS258ME-SLII was synthesized and its interactions with N-terminus and C-terminus of the N protein were demonstrated by filter-binding assay and electrophoretic mobility shift assay. Preliminary data from co-immunoprecipitation indicated a potential interaction between the viral N protein and M protein. Whether this interaction mediates assembly of the N protein-RNA genome complex into the virus particles needs to be verified. The mechanism of how viral RNA with package signal is packaged into the viral particle and the roles of structural proteins within the viral assembly needed to be further determined.