小反刍兽疫病毒与宿主细胞相互作用研究
发布时间：2022-12-24 13:40
小反刍兽疫是一种由小反刍兽疫病毒（PPRV）引起的家养和野生小反刍动物高度传染性疾病。有关PPRV复制的分子机制及其与宿主的相互作用研究仍不清楚。本研究为了更好地理解PPRV复制的分子机制及病毒与宿主细胞相互作用。利用分子生物学技术,研究PPRV感染对Vero细胞的影响,以及PPRV抵抗宿主细胞免疫的途径。我们利用MOI为3的PPRV感染Vero细胞,对感染后36h、48h、60h及72h的样本进行分析,结果表明PPRV对eIF2α具有去磷酸化的作用,在哺乳动物细胞（如HEK293T）过表达PPRV P蛋白能够诱导宿主细胞生长阻滞DNA损伤蛋白（GADD34）的表达,已知GADD34与eIF2α去磷酸化有关。此外,我们研究发现PPRV P蛋白能够抑制PERK/eIF2α磷酸化,使用eIF2α去磷酸化选择性抑制剂（Salubrinal）处理细胞,能够在蛋白水平和mRNA水平抑制PPRV复制。我们推测eIF2α去磷酸化有利于PPRV复制,而且PPRV P蛋白参与调控这一分子机制。在病毒感染的过程中,宿主细胞特有的信号通路未折叠蛋白反应（UPR）可能被激活或抑制,以对抗病毒诱导的内质网应激维...
【文章页数】：91 页
【学位级别】：博士
【文章目录】：
摘要
abstract
List of Abbreviations
ChapterⅠLiterature Review
1.1 Description and the causative agent of peste des petits ruminants
1.1.1 The PPRV virion structure and genome
1.1.2 PPRV structural proteins
1.1.3 PPRV hosts range and susceptibility
1.1.4 Pathogenesis and clinical symptoms of PPRV
1.1.5 Diagnosis of PPRV
1.1.6 Epidemiology,geographic distribution and eradication program of PPRV
1.1.7 Molecular biology and Virus/host interaction of PPRV
1.2 The aims of this study
Chapter Ⅱ Case-study of epidemiology and molecular characterization of PPRV in an outbreak in Burundi(2017-2018)
2.1 Introduction
2.2 Materials and methods
2.2.1 Suspicion of PPR outbreak in Burundi and sampling for laboratory diagnosis
2.2.2 Serology for the detection of antibodies directed against PPR virus
2.2.3 Reverse transcriptase-PCR for detection of morbillivirus and PPRV RNAs
2.2.4 Sequencing and phylogenetic analysis
2.3 Results
2.3.1 Serological and PCR analysis
2.3.2 Results of a retroactive study
2.3.3 Phylogenetic analysis
2.4 Discussion
2.5 Conclusion
Chapter Ⅲ Study of the role of PPRV phosphoprotein in virus replication and virus/host cells interaction
3.1 Introduction
3.2 Materials and Methods
3.2.1 Plasmids
3.2.2 Primers design for cloning N and P
3.2.3 PCR amplification
3.2.4 Gel electrophoresis and DNA purification
3.2.5 Restriction enzyme digestion
3.2.6 DNA ligation
3.2.7 E.coli transformation
3.2.8 Plasmid DNA extraction
3.2.9 Screening of the DNA inserts
3.2.10 Sequencing and sequences analysis
3.2.11 Cells culture,virus,antibodies and reagents
3.2.12 Transfection and transient expression of target proteins
3.2.13 SDS-PAGE and Western blotting
3.2.14 Chemical treatment
3.2.15 Immunofluorescence assay(IFA)
3.2.16 RT-q PCR assay and PPRV m RNA quantification
3.2.17 Data analysis
3.3 Results
3.3.1 Confirmation of PPRV P and PPRV N plasmids construction
3.3.2 Analysis of PPRV P and PPRV N proteins expression
3.3.3 PPRV infection represses PERK/e IF2αphosphorylation in Vero cells
3.3.4 PPRV infection modulates GADD34 protein expression in infected Vero cells
3.3.5 PPRV infection represses ER stress-induced e IF2αphosphorylation
3.3.6 Manipulation of e IF2αphosphorylation differentially regulates PPRV replication
3.3.7 PPRV P protein is involved in PPRV mediated e IF2αdephosphorylation
3.3.8 PPRV P protein induces host cellular GADD34
3.4 Discussion
3.5 Conclusion
Chapter Ⅳ PPRV modulation of UPR and Stress Granules formation
4.1 Introduction
4.2 Materials and methods
4.2.1 Plasmids
4.2.2 Cells culture,virus,antibodies and reagents
4.2.3 Transfection and transient expression of target proteins
4.2.4 Stress granule induction and assessment
4.2.5 SDS-PAGE and Western blotting
4.2.6 Immunofluorescence assay(IFA)
4.2.7 Co-immunoprecipitation(co-IP)
4.2.8 Data analysis
4.3 Results
4.3.1 PPRV infection induces ATF6 but not IRE1/XBP1 arm of the UPR
4.3.2 PPRV infection inhibits stress granules formation
4.3.3 PPRV inhibition of SGs assembly may target G3BP1
4.3.4 P protein is involved in the control of UPR
4.3.5 P protein is involved in the control of SGs formation
4.3.6 P protein alone can inhibit oxidative SG formation
4.3.7 PPRV P protein may target G3BP1 to control SG formation
4.4 Discussion
4.5 Conclusion
Chapter Ⅴ General Conclusions
REFERENCES
Acknowledgements
Author’s Curriculum Vitae
【参考文献】：
期刊论文
[1]Reverse Genetics for Peste des Petits Ruminants Virus: Current Status and Lessons to Learn from Other Non-segmented Negative-Sense RNA Viruses[J]. Alfred Niyokwishimira,Yongxi Dou,Bang Qian,Prajapati Meera,Zhidong Zhang. Virologica Sinica. 2018(06)
本文编号：3726253
【文章页数】：91 页
【学位级别】：博士
【文章目录】：
摘要
abstract
List of Abbreviations
ChapterⅠLiterature Review
1.1 Description and the causative agent of peste des petits ruminants
1.1.1 The PPRV virion structure and genome
1.1.2 PPRV structural proteins
1.1.3 PPRV hosts range and susceptibility
1.1.4 Pathogenesis and clinical symptoms of PPRV
1.1.5 Diagnosis of PPRV
1.1.6 Epidemiology,geographic distribution and eradication program of PPRV
1.1.7 Molecular biology and Virus/host interaction of PPRV
1.2 The aims of this study
Chapter Ⅱ Case-study of epidemiology and molecular characterization of PPRV in an outbreak in Burundi(2017-2018)
2.1 Introduction
2.2 Materials and methods
2.2.1 Suspicion of PPR outbreak in Burundi and sampling for laboratory diagnosis
2.2.2 Serology for the detection of antibodies directed against PPR virus
2.2.3 Reverse transcriptase-PCR for detection of morbillivirus and PPRV RNAs
2.2.4 Sequencing and phylogenetic analysis
2.3 Results
2.3.1 Serological and PCR analysis
2.3.2 Results of a retroactive study
2.3.3 Phylogenetic analysis
2.4 Discussion
2.5 Conclusion
Chapter Ⅲ Study of the role of PPRV phosphoprotein in virus replication and virus/host cells interaction
3.1 Introduction
3.2 Materials and Methods
3.2.1 Plasmids
3.2.2 Primers design for cloning N and P
3.2.3 PCR amplification
3.2.4 Gel electrophoresis and DNA purification
3.2.5 Restriction enzyme digestion
3.2.6 DNA ligation
3.2.7 E.coli transformation
3.2.8 Plasmid DNA extraction
3.2.9 Screening of the DNA inserts
3.2.10 Sequencing and sequences analysis
3.2.11 Cells culture,virus,antibodies and reagents
3.2.12 Transfection and transient expression of target proteins
3.2.13 SDS-PAGE and Western blotting
3.2.14 Chemical treatment
3.2.15 Immunofluorescence assay(IFA)
3.2.16 RT-q PCR assay and PPRV m RNA quantification
3.2.17 Data analysis
3.3 Results
3.3.1 Confirmation of PPRV P and PPRV N plasmids construction
3.3.2 Analysis of PPRV P and PPRV N proteins expression
3.3.3 PPRV infection represses PERK/e IF2αphosphorylation in Vero cells
3.3.4 PPRV infection modulates GADD34 protein expression in infected Vero cells
3.3.5 PPRV infection represses ER stress-induced e IF2αphosphorylation
3.3.6 Manipulation of e IF2αphosphorylation differentially regulates PPRV replication
3.3.7 PPRV P protein is involved in PPRV mediated e IF2αdephosphorylation
3.3.8 PPRV P protein induces host cellular GADD34
3.4 Discussion
3.5 Conclusion
Chapter Ⅳ PPRV modulation of UPR and Stress Granules formation
4.1 Introduction
4.2 Materials and methods
4.2.1 Plasmids
4.2.2 Cells culture,virus,antibodies and reagents
4.2.3 Transfection and transient expression of target proteins
4.2.4 Stress granule induction and assessment
4.2.5 SDS-PAGE and Western blotting
4.2.6 Immunofluorescence assay(IFA)
4.2.7 Co-immunoprecipitation(co-IP)
4.2.8 Data analysis
4.3 Results
4.3.1 PPRV infection induces ATF6 but not IRE1/XBP1 arm of the UPR
4.3.2 PPRV infection inhibits stress granules formation
4.3.3 PPRV inhibition of SGs assembly may target G3BP1
4.3.4 P protein is involved in the control of UPR
4.3.5 P protein is involved in the control of SGs formation
4.3.6 P protein alone can inhibit oxidative SG formation
4.3.7 PPRV P protein may target G3BP1 to control SG formation
4.4 Discussion
4.5 Conclusion
Chapter Ⅴ General Conclusions
REFERENCES
Acknowledgements
Author’s Curriculum Vitae
【参考文献】：
期刊论文
[1]Reverse Genetics for Peste des Petits Ruminants Virus: Current Status and Lessons to Learn from Other Non-segmented Negative-Sense RNA Viruses[J]. Alfred Niyokwishimira,Yongxi Dou,Bang Qian,Prajapati Meera,Zhidong Zhang. Virologica Sinica. 2018(06)
本文编号：3726253
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