- 學位論文
嚴重急性呼吸道症候群相關冠狀病毒之組裝
Viral Assembly of Severe Acute Respiratory Syndrome Associated Coronavirus
摘要
嚴重急性呼吸道症候群(Severe Acute Respiratory Syndrome，SARS)，在2002年末至2003年春天襲捲了全球32個國家，並造成將近千人死亡。經由眾多科學家的努力，在SARS病患以及和臨床檢體一起培養的African green monkey kidney cells，Vero E6中，成功地分離出一株新型的冠狀病毒(coronavirus)，命名為SARS-CoV，同時利用猴子模式證實此為引起SARS的元兇。一般所熟知的冠狀病毒為含有套膜的病毒，其基因體為一單股正向的RNA，長約30,000個核苷酸(nucleotides，nt)，是所有RNA病毒中最大的，此RNA 基因體可表達病毒複製時所需的許多蛋白質。依血清型(serotype)可以分為3個groups，其代表有human coronavirus (HCoV-229E，group 1)、mouse hepatitis virus (MHV，group 2)和infectious bronchitis virus (IBV，group 3)，感染生物體後會引起呼吸道、腸道、肝或中樞神經相關的疾病。 先前對其他型的冠狀病毒的研究指出，完整的冠狀病毒在組裝的過程時，需要spike (S)、membrane (M)、envelope (E) 和nucleocapsid (N)四個結構蛋白質的參與，在MHV和IBV中甚至只要M和E 兩個蛋白質即可製造出其類病毒顆粒(virus-like particles，VLPs)，但沒有S蛋白質則無感染力。另外有研究指出，在MHV及IBV中，各自的N protein會和基因體RNA上，靠近open reading frame (ORF) 1b 3’端的一段序列結合，形成特殊的nucleocapsid結構，進而將病毒基因體RNA帶至新組裝的病毒顆粒裡。同時，科學家也證明即使在這段RNA前面置換成非病毒的RNA，此段artificial RNA仍然可被帶入到新組裝的病毒顆粒內。因此，將此段RNA序列稱為包裹訊號(packaging signal，PS)，長度依不同型的冠狀病毒而異，核心包裹訊號約為70 nt長。 本篇論文利用可以培養SARS-CoV的Vero E6 cells，同時轉染入可以表現SARS-CoV的S、M、E和N 四個結構蛋白質的質體，經四天轉染後，將培養液以sucrose cushion centrifugation的方式分離，再配合Western blotting的分析，證明可以收集到SARS-CoV的類病毒顆粒。之後將可能的包裹訊號包含579 nt構築到能表現GFP質體之GFP基因的3’ non-coding region (NCR)，再和上述四個結構蛋白質一起在Vero E6 cells中表現，用上述同樣的方法可收集到類病毒顆粒，再感染新的Vero E6 cells後，在螢光顯微鏡下可以觀察到綠色螢光，Western blotting亦偵測到GFP的表現。對老鼠之支氣管細胞進行感染，也發現能被此SARS-CoV類病毒顆粒所感染而發出螢光。可是當製備類病毒顆粒的過程時，缺少N protein則會影響類病毒顆粒包裹RNA的能力。此結果證明我們所製備的類病毒顆粒具有感染力且構築的可能包裹訊號是具有功能的。此系統可作為研究SARS-CoV感染特定組織之有力工具。利用Northwestern blotting 和Filter binding assay發現SARS-CoV的N protein (422 a.a.)上第1~235個胺基酸和第236~384個胺基酸兩個區域會和我們所構築的SARS-CoV包裹訊號結合，然而缺少1~80或152~235個胺基酸時，皆會影響和RNA結合的能力。
並列摘要
Severe acute respiratory syndrome (SARS), an atypical pneumonia, spread out through 32 countries and took away about 800 live worldwide during the late 2002 to the spring of 2003. A novel coronavirus termed SARS-CoV was isolated from the SARS patients and Vero E6 cells inoculated with clinical specimens and was identified to be the causative agent of SARS. Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with about 30,000 nucleotides (nt) in length, which is the largest in all RNA viruses. The virus particle consists of four structural proteins: spike (S), membrane (M), envelope (E) and nucleocapsid (N). However, previous studies have demonstrates that E and M proteins are sufficient to form virus-like particles (VLPs) in MHV and IBV. The N protein of MHV and IBV can bind to a specific sequence termed packaging signal (PS) located near the 3’ terminus of the open reading frame (ORF) 1b in the genomic RNA. This facilitates the assembly of viral genomic RNA into virus particles. The length of PS varies among coronaviruses with a core domain about 70 nt. The PS is functional when cloned into the 3’ terminal region of a non-viral RNA. In this study, a plasmid containing green fluorescence protein gene and a putative PS of SARS-CoV (GFP-PS) was constructed and co-transfected with plasmids encoding the four structural proteins of SARS-CoV into Vero E6 cells that are able to support the replication of SARS-CoV. VLPs were isolated four days post-transfection from culture medium by using sucrose cushion centrifugation. Structure proteins in the SARS-CoV VLPs were detected by Western blot analysis. The VLPs were used to infect new Vero E6 cells and mouse bronchiolar cells. The green fluorescence was visualized in both cells under the fluorescence microscope and GFP in the VLPs-infected cells was detected by Western blot analysis. In addition, the GFP-PS mRNA was not packaged into the VLPs in the absence of N protein. Furthermore, Northwestern blot analysis and filter binding assay demonstrated that there are at least two RNA binding domains (amino acids 1 to 235 and 236 to 384) in the N protein of SARS-CoV. Deletions of amino acids 1 to 80 or 152 to 235 diminished the RNA binding activity. Taken together, these results indicated that the VLPs generated in this study are infectious and the putative PS is functional. The GFP-PS-containing VLPs could be used as a powerful tool to examine the tissue tropism and pathogenesis of SARS-CoV.
參考文獻
延伸閱讀
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