Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Virus, and Antibodies
2.2. Plasmid Construction
2.3. E. coli-Based Protein Expression and Purification
2.4. In Vitro Fluorescent DUB Assay
2.5. In Vitro Cleavage of K48 and K64-Linked Polyubiquitin Chains
2.6. Cell-Based DUB Assay
2.7. Cell-Based Trans- and Cis-Cleavage Assay
2.8. Construction of PRRSV Mutants and Growth Kinetics Analysis
2.9. Quantitative Real-Time PCR
2.10. Measurement of Secreted TNFα
2.11. Bioinformatics Prediction
2.12. Quantitative Analysis
2.13. Statistical Analysis
3. Results
3.1. The Downstream Flanking Sequence is Critical for the Yield and Solubility of PRRSV PLP2 Protease Domain in E. coli
3.2. The In Vitro Purified PRRSV PLP2 Can Efficiently Cleave Both K63 and K48-Linked Polyubiquitin Chains Ub3-7 but Displays a Differential Activity in Converting the Respective Ubiquitin Dimers to Monomer
3.3. Identification of Residues Critical for the PLP2 DUB Activity
3.4. Differentiation of DUB Activity from Trans- and Cis-Cleavage Activity
3.5. The PLP2 Cis-Cleavage Activity is Dispensable for HP-PRRSV Strain JXwn06 Viability in Cell Culture
3.6. HP-PRRSV-Induced Production of TNF-α and IL-1β is Strongly Associated with nsp2 that is Independent of PLP2 DUB Activity
4. Discussion
4.1. Insight into the Biochemical Properties of PRRSV PLP2
4.2. The Role of Cis-Cleavage and DUB Activities of PLP2 in PRRSV Infection
4.3. Insight into the Induction of Pro-Inflammatory Cytokine Production During PRRSV Infection
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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PLP2 Mutants | DUB Activity | Cis-Cleavage | Trans-Cleavage | Viral Viability | Cytokine Production |
---|---|---|---|---|---|
D84R | Active | √ | √ | viable | No effect |
D84N | Active | √ | √ | viable | ND |
D85R | Largely blocked | - | √ | nonviable | |
D85N | Largely blocked | - | √ | nonviable | |
T88R | Largely blocked | - | √ | nonviable | |
T88S | Active | √ | √ | viable | No effect |
T88G | Partially blocked | - | √ | viable | TNF-α, IL-1β |
E90R | Slightly blocked | √ | √ | nonviable | |
E90Q | Active | √ | √ | viable | TNF-α,IL-1β |
D91R | Largely blocked | - | √ | nonviable | |
D91N | Active | √ | √ | nonviable |
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Zhou, S.; Ge, X.; Kong, C.; Liu, T.; Liu, A.; Gao, P.; Song, J.; Zhou, L.; Guo, X.; Han, J.; et al. Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction. Viruses 2019, 11, 896. https://doi.org/10.3390/v11100896
Zhou S, Ge X, Kong C, Liu T, Liu A, Gao P, Song J, Zhou L, Guo X, Han J, et al. Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction. Viruses. 2019; 11(10):896. https://doi.org/10.3390/v11100896
Chicago/Turabian StyleZhou, Shaochuan, Xinna Ge, Can Kong, Teng Liu, Aijing Liu, Peng Gao, Jiangwei Song, Lei Zhou, Xin Guo, Jun Han, and et al. 2019. "Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction" Viruses 11, no. 10: 896. https://doi.org/10.3390/v11100896
APA StyleZhou, S., Ge, X., Kong, C., Liu, T., Liu, A., Gao, P., Song, J., Zhou, L., Guo, X., Han, J., & Yang, H. (2019). Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction. Viruses, 11(10), 896. https://doi.org/10.3390/v11100896