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研究生：	林子華 Lin, Tzu-Hua
論文名稱：	SARS 病毒ORF3 相關基因抑制蛋白質合成機制之探討 Inhibition of Protein Synthesis by ORF3 Related Genes of SARS Coronavirus
指導教授：	蕭璦莉 Shiau, Ai-Li
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	63
中文關鍵詞：	冠狀病毒、嚴重急性呼吸道症候群
外文關鍵詞：	SARS, coronavirus
相關次數：	點閱：48 下載：2
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　　嚴重急性呼吸道症候群（Severe acute respiratory syndrome, 簡稱SARS）是由一種新的冠狀病毒所引起的傳染性疾病。在SARS病毒的基因體當中的14個可能的開放讀碼區（Open reading frame, 簡稱ORF）當中，存在著許多具種別特異性的附屬蛋白。在這之中，ORF3基因被預測可以表現包括3a、3b以及SE三種蛋白，然而他們的表現對於病毒的生活史有何種意義至今仍然是不了解的。在本篇研究當中，我們建構了完整的ORF3基因序列，包括了3a、3b以及SE，並將其送入細胞內表現。實驗結果發現，ORF3基因的表現會全面性的抑制細胞內蛋白質的合成。接著，我們繼續研究ORF3基因所涵蓋的三個蛋白的表現，其個別對細胞所造成的影響。首先，我們發現3a蛋白會表現在細胞的內質網以及高基氏體上，並造成內質網緊迫(Stress)的生理反應，造成蛋白合成的抑制。而這樣的現象可能是由於3a蛋白形成了一個鈣離子通道，將內質網內儲存的鈣離子釋放所造成的結果。接著，我們發現3b蛋白的表現可能會造成細胞的死亡，因此破壞了細胞內正常的生理代謝。最後，SE蛋白則可能形成鈉離子的通道，造成細胞內產生一種高鈉濃度環境的緊迫進而抑制蛋白質的轉譯。總結而論，本篇研究發現，ORF3基因所表現的蛋白在病毒的生活史中以及對病毒毒力的影響上，可能扮演了相當重要的角色。

　A novel coronavirus has been identified as the etiologic agent of severe acute respiratory syndrome (SARS). Among the 14 potential open reading frames (ORFs) in its genome, several group-specific accessory proteins may be encoded. SARS coronavirus (SARS-CoV) ORF3 gene is a tricistronic ORF, which expresses 3a, 3b, and small envelope (SE) proteins. Though the 3a and SE proteins are known to be expressed during SARS-CoV infection, the role of these proteins in viral pathogenesis is still unknown. In the present study, we cloned the full-length ORF3 gene, including 3a, 3b, and SE, and transfected it into several lung epithelial cell lines. We found that expression of SARS-CoV ORF3 gene caused a general down-regulation of proteins synthesis. Next, we transfected three ORF3-encoded genes individually, and found that they used different mechanisms to shutdown cellular protein synthesis. First, 3a protein can localize to ER and Golgi apparatus and induced an ER stress response, possibly by forming calcium channel to release the ER-sequestrated calcium. Second, expression of 3b protein can lead to cell death, which may therefore shut-off cell metabolism. Finally, SE protein may be a potential “viroporin” and increase the intracellular sodium concentration, thus inhibiting protein translation by this “saline stress”. In conclusion, our findings suggest that these accessory molecules of SARS-CoV may play important roles during the virus life cycle and determinate viral virulence.

Table of contents

Abstract	i
中文摘要	ii
Table of Contents	iii
List of Figures	vi
List of Appendix	viii
Abbreviations	ix
Acknowledgement	x

Introduction	1
	Severe Acute Respiratory Syndrome (SARS)	1
	Coronavirus	2
	SARS-CoV ORF3 	3
	The Interactions between Viral Protein and Host Cell	4
	Regulation of Protein Synthesis in mRNA Translation	5
	Aim of the Study	8
		
Materials and methods	9
	Cell lines	9
	Plasmids	9
	Antibodies	10
	Oligonucleotides	10

 

	Plasmid constructions	12
	Cells, transfections and luciferase assays	13
	35S-Met protein synthesis assay	13
	Cell apoptosis assay	14
	RT-PCR.	14
	3H-uridine transcriptional assay	15
	35S-Met translational assay	15
	Fluorescence microscopy.	15
	Bioinformatic analysis of 3a and SE proteins	16
	Western Blot Analysis	16
	Fluorescence measurement of [Ca2+]C	17
	Fluorescence measurement of [Na+]C	17
Results	18
	Construction of SARS-CoV ORF3 gene expression vector.	18
	Construction of SARS-CoV 3a, t3a, 3b, and SE proteins expression vectors	18
	Construction of expression vectors encoding SARS-CoV 3a-, t3a-, 3b-, and SE-EGFP fusion proteins	18
	Expression of ORF3 genes inhibited cellular protein synthesis	19
	Expression of three predicted ORF3-encoded genes in the transfected A549 cells could be detected by RT-PCR	20
	Expressions of 3a, 3b, and SE genes inhibited cellular protein synthesis at the translational level	20
	The 3a-EGFP fusion protein was localized in Golgi apparatus and ER in the transfected CHO cells	21
	The C-terminal region of 3a protein has a calcium pump-like domain	21
	The t3a-EGFP fusion protein was localized in Golgi apparatus and ER in the transfected CHO cells	22
	Expression of either 3a or t3a protein caused an increase in intracellular calcium concentration	22
	Expression of both 3a & t3a proteins caused cellular ER stress response	22
	Analysis of 3a protein predicted transmembrane α-helix structure revealed two hydrophilic appearances	23
	Expression of the 3b protein induced cell death	23
	The SE-EGFP fusion proteins had a disseminated distribution in the transfected CHO cells	24
	Expression of the SE protein increased intracellular sodium concentration	24
Discussion	25
References	33

 
List of Figures

Figure 1	Construction of SARS-CoV ORF3 expression vector	40
Figure 2	Modification of the mutation site on the ORF3 gene	41
Figure 3	Construction of SARS-CoV 3a, 3b, and SE expression vectors	42
Figure 4	Construction of truncated 3a expression vectors	43
Figure 5	Construction of SARS-CoV expression vectors encoding 3a-, 3b-, and SE-EGFP 	44
Figure 6	Inhibition of promoter activities of IL-8 and CMV, as well as transcription activity of NF-κB and AP1, by ORF3 gene expression	45
Figure 7	The expression of SARS-CoV ORF3 gene inhibits the housekeeping reporter gene activities in various human and mouse cell lines	46
Figure 8	35[S]-Met protein synthesis assay	47
Figure 9	Detection of three predicted ORF3 encoded-genes in the transfected A549 cells by using RT-PCR	48
Figure 10	Expression of 3a, 3b, and SE genes inhibited the β-actin promoter activities in A549 cells	49
Figure 11	3[H]-uridine transcriptional assay	50
Figure 12	35[S]-Met translational assay	51
Figure 13	Determination of the cellular localization of 3a-EGFP fusion protein by florescence microscopy	52
Figure 14	Determination of the cellular localization of t3a-EGFP fusion protein by florescence microscopy	53
Figure 15	Detection of intracellular calcium concentration by Fura-3 florescence calcium indicator	54
Figure 16	Detection of 3a protein-induced ER stress signaling response by western blotting	55
Figure 17	The predicted transmembrane α-helix structure of 3a protein	56
Figure 18	Hypothetical model of 3a protein-induced ER stress response	57
Figure 19	Expression of the 3b protein induced cell death	58
Figure 20	Cellular localization of SE protein and the alteration of sodium concentration	59
Figure 21	Hypothetical model of SE protein-induced “saline stress”	60

 
List of Appendix

Appendix 1	Position of ORF3 in the Genome of SARS-CoV	61
Appendix 2	Bioinformatic analysis of the amino acid sequence of the 3a protein	62
Appendix 3	Bioinformatic analysis of the amino acid sequence of the SE protein	63
                                    

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校內：2007-08-29公開
校外：2007-08-29公開

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