I.家禽里奧病毒感染細胞之蛋白質體學分析以鑑定參與病毒感染機制之可能蛋白質 II.外源性雞細胞素白細胞介素1β促進家禽里奧病毒對宿主細胞之感染

Part I：Proteomics Analysis of Avian Reovirus Infected Host Cells to Identify Candidate Proteins Involved in Virus Infection Mechanisms. Part II：Exogenous Chicken Cytokine Interleukin-1β Enhanced Avian Reovirus Infection in Host Cells.

吳以樂(Yi-Le Wu)

指導教授 ： 殷献生

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2010年)

摘要

I.家禽里奧病毒感染細胞之蛋白質體學分析以鑑定參與病毒感染機制之可能蛋白質
家禽里奧病毒(Avian reovirus)屬於里奧病毒科(Reoviridae family)，是非封套核醣核酸病毒(non-enveloped RNA viruse)，具有十條雙股核醣核酸組成其基因體。此類病毒是鳥類重要病源，其感染主要經由糞口傳染途徑，在家禽業中造成可觀的經濟損失。因此，更多地了解病毒感染機制可造福家禽業。蛋白質體學分析是鑑定家禽里奧病毒感染前後宿主細胞蛋白質含量差異全面情形的良好技術。為了瞭解家禽里奧病毒對宿主細胞調控的整體情形，首先以免疫螢光顯微染色方式鑑定家禽里奧病毒感染所造成的細胞股價結構差異，並且以MTT和AlamarBlue檢驗方式確認在感染過程中的細胞活性。2D-DIGE以及其後續的MALDI-TOF MS分析用以鑑定在家禽里奧病毒感染過程中被正調控或負調控的蛋白質，依照不同的功能和細胞內分布區域將這些蛋白質分類，並且以西方墨點法確認2D-DIGE的結果。免疫螢光染色的結果顯示細胞內肌動蛋白(actin)的總量雖然不變但是排列在家禽里奧病毒感染後被改變。AlamarBlue檢測確認在感染過程中不變的細胞活性。2D-DIGE與MALDI-TOF MS檢測鑑定出八十七個在家禽里奧病毒感染過程中表現量受到調控的蛋白質。在這些蛋白質中，PRDX6 (peroxiredoxin 6)、PGAM1 (Phosphoglycerate mutase 1)、PSME3 (Proteasome activator complex subunit 3)、ANXA5 (Annexin A5)、PRDX4 (peroxiredoxin 4)、HSP60 (60 kDa heat shock protein)、SUMO3 (Small ubiquitin-related modifier 3)、VDAC2 (Voltage-dependent anion channel 2)等利用西方墨點法進行確認。這些蛋白質的功能以及在感染過程中的可能的調控機制皆被討論。我們進行了首次對於受家禽里奧病毒感染的雞纖維芽母細胞(chicken fibroblast embryo cells)進行的蛋白質體學測試，所鑑定出表現量有差異的蛋白質所參與的細胞功能包括細胞骨架組成、細胞凋零調控、能量代謝、訊息傳遞以及泛素蛋白酶體途徑(ubiquitin-proteasome pathway)，這些數據提供了解家禽里奧病毒感染過程的基本了解，並且促進病毒分子致病研究中可能蛋白質的標定。

II.外源性雞細胞素白細胞介素1β促進家禽里奧病毒對宿主細胞之感染
促炎細胞因子(pro-inflammatory cytokine) 白細胞介素1β(interleukin-1β)是白細胞介素家族的一員，屬於受病毒感染之細胞主要抗病毒反應之一。白細胞介素1β所引發的訊息傳遞途徑包括吸引MyD88與IL-1RAcP相結合，以及下游活化IRAKs和TRAF6等蛋白質，最終促進NF-κB與AP-1等轉錄因子(transcription factors)的活化。由於在被培養的細胞受家禽里奧病毒感染後之培養上清液中，白細胞介素1β的活性被發現有加強的現象，並且，白細胞介素1β過度表現(overexpression)相連的關節炎徵狀是家禽里奧病毒S1133病毒株感染雞隻後的主要反應，推測在家禽里奧病毒感染過程中白細胞介素1β可能扮演著重要角色。本實驗發現，在對於培養的雞細胞施與外源性雞細胞素白細胞介素1β時，家禽里奧病毒的感染有增強的情形。當細胞先被施與雞白細胞介素1β再受到家禽里奧病毒的感染時，病毒力價、病毒蛋白與核醣核酸量都提高，並且這樣的提升是決定於外加白細胞介素1β量。將白細胞介素1β途徑下游蛋白質MyD88過表現，更進一步地確認白細胞介素1β對於病毒感染的促進效果。白細胞介素1β病毒感染促進效果之可能應用亦被討論。

關鍵字

家禽里奧病毒 ； 蛋白質體學 ； 雞細胞素白細胞介素1 ； 病毒感染 ； 2D-DIGE

並列摘要

Part I: Proteomics Analysis of Avian Reovirus Infected Host Cells to Identify Candidate Proteins Involved in Virus Infection Mechanisms.
Avian reoviruses (ARVs) are members of Reoviridae family, which are non-enveloped RNA viruses with 10 double-stranded RNA as genome content. The viruses are important pathogens of birds that are mainly transported through fecal-oral route, causing considerable economic losses in the poultry industry, and thus understanding more about the viral infection mechanism would be beneficial to the poultry industry. Proteomic analysis is a good technique to identify a whole view of the protein quantity differences in the host cell pre- and post-infection by ARV.
To acquire the whole regulatory view of ARV to the host cell, immunofluorescence microscopy was first used to identify the cytoskeleton structure differences caused by ARV infection. The MTT and AlamarBlue assays were performed to verify the cell viability during the infection process. The 2D-DIGE, followed by MALDI-TOF MS analysis, was then used to identify the proteins up- or down-regulated during the ARV infection process. These proteins were classified by different functions and locations. Validation of 2D-DIGE was carried out by Western blot analysis.
The immunofluorescence microscopy showed the arrangement, but not the quantity of cytosol F-actin, was changed after ARV infection. The AlamarBlue assay assured the unchanged cell viability. The 2D-DIGE and MALDI-TOF MS analysis identified 87 proteins with expression level regulated during ARV infection process. Among these proteins, peroxiredoxin 6 (PRDX6), Phosphoglycerate mutase 1 (PGAM1), Proteasome activator complex subunit 3 (PSME3), Annexin A5 (ANXA5), peroxiredoxin 4 (PRDX4), 60 kDa heat shock protein (HSP60), Small ubiquitin-related modifier 3 (SUMO3), and Voltage-dependent anion channel 2 (VDAC2) were used in the validation performed with Western blot analysis. The functions of these proteins and the possible regulatory mechanism in the infection process were then being discussed.
We performed the first analysis of the proteome of chicken fibroblast embryo cells (DF-1) infected with avian reovirus. A series of differentially expressed cellular proteins associated with cytoskeleton components, regulators of apoptosis, energy metabolism, signal transduction, and ubiquitin-proteasome pathways were identified. These data provides the fundamental understanding of processes during ARV infection and facilitates probing the candidates for viral molecular pathogenesis studies.

Part II:Exogenous Chicken Cytokine Interleukin-1βEnhanced Avian Reovirus Infection in Host Cells.
The pro-inflammatory cytokine Interleukin-1β (IL-1β) is one of the members of IL-1 family and is belonged to the primary antiviral responses in virus-infected cells. The signal transduction pathway caused by IL-1β involved the recruitment of MyD88 to the IL-1RAcP and the down-stream activation of IRAKs and TRAF6 proteins, which would finally induce the activation of transcription factors such as NF-κB and AP-1. Because the enhanced IL-1β activity was found in culture supernatant of avian reovirus (ARV) infected cell and the IL-1β over expression related symptom arthritis syndrome is the primary effect in ARV strain S1133 infected chicken, it is implied that the chicken IL-1βmay play an important role on ARV infection processes. In this study, we found that ARV infection was enhanced through treatment of exogenous chicken cytokine IL-1β to the cultured chicken cells. The increases of viral titers, viral protein quantities and viral RNA quantities were found while the cells were pretreated by chicken IL-1β before infected by ARV, and the level of increases were depend upon the quantities of added exogenous IL-1β proteins. Overexpression of the down-stream adopter protein MyD88 further proved the IL-1β enhancement. The possible applications of the infection-regulatory role of IL-1β were also discussed.

並列關鍵字

Avian Reovirus ； Proteomics ； Chicken Cytokine Interleukin-1 ； Virus Infection

參考文獻

2. Jones RC (2000.) Avian reovirus infections. Rev. Sci.Tech. 19:614-625.

3. van der Heide L (2000) The history of avian reovirus. Avian Dis. 44:638-641.

4. Grande A, Rodriguez, E., Costas, C., Everitt, E., Benavente, J. (2000) Oligomerization and cell-binding properties of the avian reovirus cell-attachment protein sigmaC. Virology 274:367-377.

5. O'Hara D PM, Cepica D, Coombs KM, Duncan R. (2001) Avian reovirus major mu-class outer capsid protein influences efficiency of productive macrophage infection in a virus strain-specific manner. J Virol. 75(11):5027-5035.

6. Hsien Sheng Yin J-HS, and Long Huw Lee (2000) Synthesis in Escherichia coli of Avian Reovirus Core Protein σA and Its dsRNA-Binding Activity. Virology 266:33-41.