The Innate Immune DNA Sensing cGAS-STING Signaling Pathway Mediates Anti-PRRSV Function
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Virus and Reagents
2.2. Establishment of Marc-145 Cell Line Stably Expressing Porcine STING
2.3. Construction of Recombinant HP-PRRSV XJ17-5 Strain Carrying dsRed
2.4. CRISPR gRNA Design and Preparation of STING−/− Marc-145 Cells
2.5. Porcine CD163 Gene Cloning and Establishment of Porcine Macrophages Stably Expressing CD163 for PRRSV Infection
2.6. Porcine STING RNA Interference by siRNA
2.7. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)
2.8. Western Blot Analysis
2.9. Flow Cytometry
2.10. Virus TCID50 Titration
2.11. Statistical Analysis
3. Results
3.1. Stable Expression of Porcine STING Suppresses PRRSV Infection in Marc-145 Cells
3.2. The Endogenous STING in Marc-145 Cells Mediates Antiviral Activity against PRRSV
3.3. Both Ectopic STING and Endogenous STING Inhibit PRRSV Replication in Porcine Macrophages
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Names | Primer Sequences (5′-3′) * |
---|---|
XJ17-5-PacI-F1 XJ17-5 -AflII-R1 XJ17-5 -AflII-F2 XJ17-5 -AscI-R2 XJ17-5 -AscI-F3 XJ17-5 -1R3 XJ17-5 -NotI-2R3 dsRed- KpnI-F dsRed- BclI-R | AGCTCGTTAATTAATACATGACGTATAGGTGTTGGCT CATAGGTGCTTAAGTTCATTACCACCTGTAACGGAT ATCCGTTACAGGTGGTAATGAACTTAAGCACCTATG CCTTTCTGGCGCGCCCGAAAC GTTTCGGGCGCGCCAGAAAGG AGCGAGGAGGCTGGGACCATGCCGGCCTTTTTTTTT TTTTTTTTTTTTAATTACGGCCGCATGGTTCT ACAGGCGGCCGCGTCCCATTCGCCATTACCGAGGGG ACGGTCCCCTCGGAATGTTGCCCAGCCGGCGCCAGC GAGGAGGCTGGGACCAT# ATTGAAGGTACCGCCACCATGGCCTCCTCCGAGGA TGCCGCGGAATGATCACTACAGGAACAGGTGGTGGC |
gRNA Names | gRNA Encoding DNA Sequences (5′-3′) |
---|---|
STING gRNA1-F STING gRNA1-R STING gRNA2-F STING gRNA2-R STING gRNA-PCR-F STING gRNA-PCR-R | CACCGTGGATGGATGCAGACTGGAG AAACCTCCAGTCTGCATCCATCCAC CACCGCCATCCATCCCGTGTCCCAG AAACCTGGGACACGGGATGGATGGC PCR Primer Sequences TCGCAGAGACAGGAGCTTTG GGCTGCAGACCCCATTTAAC |
siRNA Name | Primer Sequences (5′-3′) |
---|---|
siSTING-11-F siSTING-11-R siSTING- 529-F siSTING- 529-R siSTING-689-F siSTING-689-R siNC-F siNC-R | CCAGCCUGCAUCCAUCCAUTT AUGGAUGGAUGCAGGCUGGTT GCUCGGAUCCAAGCUUAUATT UAUAAGCUUGGAUCCGAGCTT CCGACCGUGCUGGCAUCAATT UUGAUGCCAGCACGGUCGGTT UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT |
Primer Names | Primer Sequences (5′-3′) |
---|---|
PRRSV Npro-F PRRSV Npro-R S-β-actin-F S-β-actin-R | ATAACAACGGCAAGCAGCAG CTCTGGACTGGTTTTGTTG ATGAAGATCAAGATCATCGCG TCGTACTCCTGCTTGCTGATC |
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Xu, Y.; Zhang, Y.; Sun, S.; Luo, J.; Jiang, S.; Zhang, J.; Liu, X.; Shao, Q.; Cao, Q.; Zheng, W.; et al. The Innate Immune DNA Sensing cGAS-STING Signaling Pathway Mediates Anti-PRRSV Function. Viruses 2021, 13, 1829. https://doi.org/10.3390/v13091829
Xu Y, Zhang Y, Sun S, Luo J, Jiang S, Zhang J, Liu X, Shao Q, Cao Q, Zheng W, et al. The Innate Immune DNA Sensing cGAS-STING Signaling Pathway Mediates Anti-PRRSV Function. Viruses. 2021; 13(9):1829. https://doi.org/10.3390/v13091829
Chicago/Turabian StyleXu, Yulin, Youwen Zhang, Shaohua Sun, Jia Luo, Sen Jiang, Jiajia Zhang, Xueliang Liu, Qi Shao, Qi Cao, Wanglong Zheng, and et al. 2021. "The Innate Immune DNA Sensing cGAS-STING Signaling Pathway Mediates Anti-PRRSV Function" Viruses 13, no. 9: 1829. https://doi.org/10.3390/v13091829