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The modulation of host immune responses by porcine circovirus type 2 ORF2 and ORF3

  • 최창용 (대학원 생명공학과 동물생명공학전공)

초록/요약

Porcine circovirus type 2 (PCV2) is the main etiological agent of postweaning multisystemic wasting syndrome (PMWS). The mechanism of pathogenicity associated with PCV2 infection is still not fully understood. Nevertheless, the fact that large amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection may induce chronic inflammatory responses followed by the destruction of lymphoid tissues. However, how PCV2 infection causes an excessive inflammatory response in the host immune system during the early stage of PCV2 infection is still not elucidated. Defining how each ORF of PCV2 manipulates the host immune system may be helpful to understand disease progression of PMWS. In this study, it was found that PCV2 ORF3 directly interact with RGS16 in the cytoplasm of host epithelial cells and its interaction leads to ubiquitin-mediated proteasomal degradation of RGS16. Facilitated degradation of the RGS16 by PCV2 ORF3 further enhances NFB translocation into the nucleus through the ERK 1/2 signaling pathway and increased secretion of IL-6 and IL-8 proinflammatory cytokine. Consequently, more severe inflammatory responses and leukocyte infiltration occur around PCV2 infection site. Furthermore, direct interaction between the PCV2 ORF2 and C1QBP within the cytoplasm of host macrophages was demonstrated in this study. The physical interaction between PCV2 ORF2 and C1QBP inhibits ubiquitin-mediated proteasomal degradation of C1QBP in macrophages. Increased stability of the C1QBP by the interaction with PCV2 ORF2 further enhances phagocytic activity of porcine macrophages through the phosphoinositol-3-kinase (PI3K) signaling pathway. These results may provide the first evidence showing how PCV2 infection facilitates the expression of proinflammatory cytokines, enhances phagocytic activity of macrophages and creates a more severe inflammatory response during the early stages of PCV2 infection.

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Contents
Abstract ii
Contents iii
List of Figures viii
List of Table x

Chapter 1. Literature review 1

1-1. The porcine circovirus and PMWS 1
1-2. The viral genome and proteins of PCV2 5
1-3. Regulator of G protein signaling 16 and immune responses 9
1-4. Complement system, C1QBP and phagocytosis 12
Reference 18

Chapter 2. The ORF3 protein of porcine circovirus type 2 promotes secretion of IL-6 and IL-8 in porcine epithelial cells by facilitating proteasomal degradation of regulator of G protein signaling 16 through physical interaction 29

2-1. Introduction 29

2-2. materials and Methods 32
2-2-1. Reagents 32
2-2-2. Viral infection and titration 32
2-2-3. Cloning and expression of each PCV2 ORF gene 33
2-2-4. Quantitation of the interaction between RGS16 and PCV2 ORF3 in yeast two-hybrid system 34
2-2-5. Confocal microscopy, western blot analyses, co-IP and ubiquitination assays 35
2-2-6. Measurement of IL-6 and IL-8 expression and neutrophil transwell migration assay 35
2-2-7. Statistical analysis 36

2-3. Results 38
2-3-1. The PCV2 ORF3 physically interacts with the N-terminal of host RGS16 within cytoplasm 38
2-3-2. Interaction between PCV2 ORF3 and host RGS16 facilitates ubiquitin-mediated proteasomal degradation of host RGS16 43
2-3-3. Decreased expression of RGS16 enhances NFkB translocation into the nucleus through the ERK1/2 signaling pathway in host cells 53
2-3-4. Increased IL-6 and IL-8 expression in PCV2 infected cells leads to increased migration of neutrophils to host cells 61

2-4. Discussion 68

Reference 73

Chapter 3. The ORF2 protein of porcine circovirus type 2 promotes phagocytic activity of porcine macrophages by inhibiting proteasomal degradation of complement component 1, q subcomponent binding protein (C1QBP) through physical interaction 81

3-1. Introduction 81

3-2. Materials and Methods 83
3-2-1. Antibodies and reagents 83
3-2-2. Cells and viruses 84
3-2-3. Viral infection and titration 84
3-2-4. Generation of cDNA sequence and gene expression analyses of pig C1qbp 85
3-2-5. Phylogenetic analysis 86
3-2-6. Overexpression of pig C1QBP in 3D4/31 cells 87
3-2-7. Western blot analyses 88
3-2-8. Flow cytometry analyses 88
3-2-9. Histology and immunohistochemistry analyses 89
3-2-10. Cloning and expression of each PCV2 ORF gene 89
3-2-11. Generation of ORF2-Flag and ORF2-EGFP expressing 3D4/31 cells 90
3-2-12. Quantitative real-time PCR analyses 91
3-2-13. Co-IP 92
3-2-14. Confocal microscopy 93
3-2-15. Measurement of C1QBP protein stability 94
3-2-16. Ubiquitination assays 94
3-2-17. Measurement of phagocytic activity 94
3-2-18. Inhibition of C1QBP expression 95
3-2-19. Measurement of IL-6 and IL-8 secretion 96
3-2-20. Statistical analyses 96

3-3. Results 97
3-3-1. cDNA cloning and expression analyses of pig C1QBP 97
3-3-2. The PCV2 ORF2 interacts with C1QBP within cytoplasm and increases C1QBP protein 103
3-3-3. Interaction between PCV2 ORF2 and C1QBP increases the stability of C1QBP by inhibiting ubiquitin-mediated proteasomal degradation 110
3-3-4. Increased stability of C1QBP by the interaction with PCV2 ORF2 enhances the phagocytic activity of host macrophages via the phosphoinositide 3-kinase signalling pathway. 116

3-4. Discussion 124

Reference 126

요 약 132

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