臺灣博碩士論文加值系統

2019年爆發的新冠病毒(Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2)對全球健康及經濟造成重大衝擊。感染後病程嚴重程度因人而異，但病毒如何與宿主免疫系統互動之機制尚未明確。先前研究指出，重症患者的第一型干擾素(Type I Interferon, IFN-)表現與功能普遍下降，顯示病毒可能干擾宿主先天免疫反應。
新冠病毒編碼的非結構蛋白中包含3-胰凝乳蛋白酶樣蛋白酶(3-chymotrypsin-like protease, 3CLp)，可切割聚蛋白以促進病毒複製。有研究推測該蛋白酶可能會切割在第一型干擾素訊號傳遞中扮演關鍵角色的白細胞介素1受體相關激酶1(Interleukin 1 receptor associated kinase 1, IRAK1)，並可能進一步作用於下游抗病毒蛋白寡腺苷酸合成酶1(Oligoadenylate synthase 1, OAS1)，但其具體情況及影響仍待證實。
本研究以人類胚胎腎細胞(HEK293T)為模型，透過質體轉染或慢病毒載體表達3-胰凝乳蛋白酶樣蛋白酶，並觀察其對白細胞介素1受體相關激酶1與寡腺苷酸合成酶1之影響。隨後以Poly I:C或R837模擬先天性免疫刺激，並檢視第一型干擾素及其下游干擾素刺激基因(Interferon-stimulated genes, ISGs)之表達狀況。實驗結果顯示，3-胰凝乳蛋白酶樣蛋白酶能裂解介白素1受體相關激酶1並抑制第一型干擾素表達，亦能切割寡腺苷酸合成酶1，可能削弱其抗病毒功能。此外，透過免疫螢光染色觀察發現，寡腺苷酸合成酶1定位也因蛋白酶作用而改變。
綜合結果顯示，新冠病毒3-胰凝乳蛋白酶樣蛋白酶具有干擾第一型干擾素路徑關鍵分子之能力，可能為病毒抑制宿主先天免疫反應、導致重症之潛在機制之一。本研究有助於深入理解病毒與宿主先天免疫之交互作用，並為未來藥物開發提供潛在目標。

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 caused a major global health and economic crisis. The severity of COVID-19 progression varies among individuals, and the mechanisms by which the virus interacts with the host immune system remain incompletely understood. Previous studies have shown that patients with severe COVID-19 exhibit reduced expression and function of type I interferon (IFN-I), suggesting that the virus may suppress innate immune responses.
SARS-CoV-2 encodes a 3-chymotrypsin-like protease (3CLp) within its non-structural proteins, which is essential for viral replication by cleaving viral polyproteins. Recent evidence suggests that 3CLp may also target host immune signaling proteins, including interleukin-1 receptor-associated kinase 1 (IRAK1), which plays a pivotal role in IFN-I production. In addition, oligoadenylate synthase 1 (OAS1), a downstream IFN-stimulated antiviral effector, may also a substrate of 3CLp, though this has not been clearly validated.
I this study, we used human embryonic kidney (HEK293T) cells as a model to investigate the interaction between 3CLp and key components of the IFN-I signaling pathway. through plasmid transfection and lentiviral expression of 3CLp, combined with Poly I:C or R837 stimulation to mimic innate immune activation, we assessed protein cleavage by Western blot, mRNA expression by qPCR, and subcellular localization by immunofluorescence staining.
Our results demonstrate that SARS-CoV-2 3CLp can cleave IRAK1, leading to downregulation of IFN-I expression. Furthermore, 3CLp was shown to cleave OAS1, potentially impairing its antiviral function. Changes in OAS1 localization were also observed upon protease activity. These findings suggest that SARS-CoV-2 3CLp may contribute to immune evasion by targeting key molecules in the IFN-I pathway. This study enhances our understanding of viral-host immune interactions and may inform future antiviral drug development targeting viral protease.

Acknowledgements i
Abbreviation list ii
Content iv
Figure contents vii
中文摘要 ix
Abstract x
1. Introduction 1
1.1 SARS-CoV-2 1
1.1.1 Cell entry mechanisms of SARS-CoV-2 2
1.1.2 SARS-CoV-2 polyprotein processing 2
1.1.3 SARS-CoV-2 against the innate immune system 3
1.1.4 3CLp inhibitor in clinical practice 3
1.2 Innate immune response 4
1.2.1 Toll-like receptors 4
1.2.2 Interleukin 1 receptor kinase 1 (IRAK1) 4
1.2.3 Type I interferon (IFN-I) 5
1.2.4 Interferon-stimulated genes (ISGs) 6
1.2.5 Oligoadenylate synthase 1 (OAS1) 12
1.3 Hypothesis 13
1.4 Research aim 14
2. Materials and Methods 16
2.1 Materials 16
2.1.1 Plasmid 16
2.1.2 Cell lines 17
2.1.3 Competent cells 17
2.1.4 Primers 17
2.1.5 Antibodies 19
2.1.6 Reagent 20
2.2 Methods 23
2.2.1 Cell culture 23
2.2.2 Gene cloning 23
2.2.3 Transformation 26
2.2.4 Transfection 26
2.2.5 Lentivirus production 27
2.2.6 Transduction 28
2.2.7 R837 or Poly I:C induced IFN-I production and signaling pathway model 28
2.2.8 DNA electrophoresis 29
2.2.9 Total RNA isolation 29
2.2.10 Reverse transcription (RT) 29
2.2.11 Real-time quantitative PCR (RT-qPCR) analysis 30
2.2.12 Western Blotting (WB) analysis 30
2.2.13 Immunofluorescence staining analysis 31
2.2.14 Statistical analysis 32
3. Results 33
3.1 Prediction of the potential cleavage sites of the IFN-I production pathway by SARS-CoV-2 3CLp 33
3.2 SARS-CoV-2 3CLp can cleave the predicted specific peptide of IRAK1 in IFN-I production pathway 34
3.3 The cleavage of IRAK1 requires the enzymatic activity of SARS-CoV-2 3CLp 34
3.4 SARS-CoV-2 3CLp can cleave IRAK1 at Q457 35
3.5 SARS-CoV-2 3CLp can suppress IFN-I expression 36
3.6 SARS-CoV-2 3CLp can suppress ISGs expression 37
3.7 SARS-CoV-2 3CLp can cleave OAS1 in IFN-I signaling pathway 39
3.8 SARS-CoV-2 3CLp can cleave OAS1 at Q384 40
3.9 The cleavage of OAS1 p46 by SARS-CoV-2 3CLp requires enzymatic activity 41
3.10 SARS-CoV-2 3CLp can alter the distribution of OAS1 p46 to cytoplasm 41
4. Discussion 43
5. Conclusion 47
6. References 49
7. Figures 60
Figure 1. The specific peptide of the cleavage by SARS-CoV-2 3CLp. 60
Figure 2. The prediction of cleavage in IRAK1 by SARS-CoV-2 3CLp. 62
Figure 3. SARS-CoV-2 3CLp can cleave IRAK1 as prediction. 63
Figure 4. The cleavage of IRAK1 requires the enzymatic activity of SARS-CoV-2 3CLp. 65
Figure 5. Nirmatrelvir can suppress the cleavage or IRAK1 by SARS-CoV-2 3CLp. 66
Figure 6. SARS-CoV-2 3CLp cleaves IRAK1 at Q457. 68
Figure 7. SARS-CoV-2 3CLp suppresses the expression of type I interferon. 70
Figure 8. SARS-CoV-2 3CLp inhibits the expression of interferon-stimulated gene, induced by R837. 72
Figure 9. SARS-CoV-2 3CLp suppresses interferon-stimulated genes expression, induced by Poly I:C. 74
Figure 10. SARS-CoV-2 3CLp inhibits the expression of IFIT1 and OASL, induced by R837. 76
Figure 11. SARS-CoV-2 3CLp reduces the expression of RIG-I, ZAP, and OASL, induced by Poly I:C. 77
Figure 12. SARS-CoV-2 3CLp suppresses the expression of PKR and MX1, induced by R837. 78
Figure 13. SARS-CoV-2 3CLp suppresses the expression of ISG20, MX1, and Viperin, induced by Poly I:C. 79
Figure 14. SARS-CoV-2 3CLp reduces the expression of OAS1, OAS2, and RNaseL, induced by R837. 80
Figure 15. SARS-CoV-2 3CLp inhibits the expression of OAS1 and OAS2, induced by Poly I:C. 81
Figure 16. SARS-CoV-2 3CLp suppresses the expression of IFIT2, induced by R837. 82
Figure 17. SARS-CoV-2 3CLp inhibits the expression of IFIT3. 83
Figure 18. SARS-CoV-2 3CLp reduces the expression of IRF7 and TRIM25, induced by Polu I:C. 84
Figure 19. The prediction of cleavage in OAS1 by SARS-CoV-2 3CLp. 85
Figure 20. SARS-CoV-2 3CLp can cleave OAS1 p46 as prediction. 86
Figure 21. SARS-CoV-2 3CLp cleaves OAS1 p46 at Q384. 87
Figure 22. The cleavage of OAS1 p46 by SARS-CoV-2 3CLp was inhibited by nirmatrelvir. 89
Figure 23. The distribution of OAS1 p44 and OAS1 p46. 90
Figure 24. SARS-CoV-2 3CLp altered the localization of OAS1. 91
Figure 25. SARS-CoV-2 3CLp suppressed RNA degradation. 92
Figure 26. The consensus sequence, which cleaved by Enterovirus (EV)68 3Cp, EV71 3Cp, Norwalk virus (NV) 3CLp, SARS-CoV-1 3CLp, or SARS-CoV-1 3CLp. 93
Figure 27. Schematic diagram representing SARS-CoV-2 3CLp antagonizes type I interferon production and signaling pathway through IRAK1 and OAS1 cleavage. 94

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