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

張展華

doi:10.6342/NTU201802934

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核壳蛋白质在中东呼吸症候群冠状病毒RNA包裹讯号辨识及病毒组装中扮演的角色

Involvement of MERS-CoV nucleocapsid protein in RNA package signal recognition and virion assembly

张展华 , 硕士　　导师：张鑫　　

英文 DOI： 10.6342/NTU201802934

中东呼吸症候群冠状病毒；包裹讯号；核壳蛋白质；类病毒颗粒； Middle East respiratory syndrome coronavirus (MERS-CoV) ； RNA package signal ； nucleocapsid (N) protein ； virus-like particle (VLP)

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摘要 │ 参考文献 (72) │ 国际计量

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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.

参考文献 ( 72 ) 〈TOP〉

1. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, et al. Isolation of novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012; 367: 1814–1820.
2. de Groot RJ, Baker SC, Baric RS, Brown CS, et al. Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J Virol. 2013; 87: 7790-7792.
3. van Boheemen S, de Graaf M, Lauber C, Bestebroer TM, et al. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio. 2012; 3: e00473-12.
4. Raj VS, Osterhaus AD, Fouchier RA, and Haagmans BL. MERS: emergence of a novel human coronavirus. Curr Opin Virol. 2014; 5: 58-62.
5. WHO, Middle East respiratory syndrome coronavirus (MERS-CoV) (http://www.who.int/emergencies/mers-cov/en/)

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引用含有DOI的文献

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。 如想对DOI的使用与概念有进一步了解，请参考 （ ） 。

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。如想对DOI的使用与概念有进一步了解，请参考（）。