Screening of Porcine Innate Immune Adaptor Signaling Revealed Several Anti-PRRSV Signaling Pathways
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Virus and Reagents
2.2. Subcloning of Porcine Innate Immune Signaling Adaptor Gene ORFs
2.3. Preparation of Polyclonal Antibody against N Protein of PRRSV
2.4. Construction of Full-Length cDNA Clones of HP-PRRSV XJ17-5 Carrying EGFP
2.5. RNA Interference by siRNA
2.6. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)
2.7. Western Blot Analysis
2.8. Flow Cytometry
2.9. Virus TCID50 Titration
2.10. Statistical Analysis
3. Results
3.1. The Antiviral Activities of Ectopic Porcine Innate Signaling Adaptor Proteins against PRRSV Infection
3.2. The Antiviral Activities of Ectopic Porcine Innate Signaling Adaptor Proteins against PRRSV-EGFP Infection
3.3. The Antiviral Activities of Endogenous TRIF, MAVS and STING Signaling against PRRSV Replication
3.4. Knockdown of Endogenous TRIF, MAVS and STING Enhances PRRSV Replication
3.5. Innate TLR3-TRIF Signaling Plays a Differential Role in Marc-145 Cells and 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′) |
---|---|
PRRSV-N-F | CATGCCATGGATGCCAAATAACAACGGCAAGC |
PRRSV-N-R | CCGCTCGAGTACTGAGGGTGATGCTGTGGC |
Primer Names | Primer Sequences (5′-3′) * |
---|---|
XJ17-5 -PacI-F1 | AGCTCGTTAATTAATACATGACGTATAGGTGTTGGCT |
XJ17-5 -AflII-R1 | CATAGGTGCTTAAGTTCATTACCACCTGTAACGGAT |
XJ17-5 -AflII-F2 | ATCCGTTACAGGTGGTAATGAACTTAAGCACCTATG |
XJ17-5 -AscI-R2 | CCTTTCTGGCGCGCCCGAAAC |
XJ17-5 -AscI-F3 | GTTTCGGGCGCGCCAGAAAGG |
XJ17-5 -1R3 | AGCGAGGAGGCTGGGACCATGCCGGCCTTTTTTTTT TTTTTTTTTTTTAATTACGGCCGCATGGTTCT |
XJ17-5 -NotI-2R3 | ACAGGCGGCCGCGTCCCATTCGCCATTACCGAGGGG ACGGTCCCCTCGGAATGTTGCCCAGCCGGCGCCAGC GAGGAGGCTGGGACCAT # |
siRNA Name | Primer Sequences (5′-3′) |
---|---|
TRIF-1022-F | CCACCUCUCCAAAUACCAATT |
TRIF-1022-R | UUGGUAUUUGGAGAGGUGGTT |
TRIF-1330-F | GCGAUAGACCACUCAGCUUTT |
TRIF-1330-R | AAGCUGAGUGGUCUAUCGCTT |
TRIF-1547-F | CCCAGAUCUUCGCUAGGAATT |
TRIF-1547-R | UUCCUAGCGAAGAUCUGGGTT |
MAVS-730-F | GCAGGGUCAGUUGUAUCUATT |
MAVS-730-R | UAGAUACAACUGACCCUGCTT |
MAVS-1028-F | CCAAUCCAGCACCAUCCAATT |
MAVS-1028-R | UUGGAUGGUGCUGGAUUGGTT |
MAVS-1364-F | GCCCAGAGGAGAAUGAGUATT |
MAVS-1364-R | UACUCAUUCUCCUCUGGGCTT |
STING-374-F | GCCUUUCGCAGGCACUGAATT |
STING-374-R | UUCAGUGCCUGCGAAAGGCTT |
STING-530-F | CCCGGAUUCAAACUUACAATT |
STING-530-R | UUGUAAGUUUGAAUCCGGGTT |
STING-713-F | GGGUUUACAGCAACAGCAUTT |
STING-713-R | AUGCUGUUGCUGUAAACCCTT |
NC-F | UUCUCCGAACGUGUCACG UTT |
NC-R | ACGUGACACGUUCGGAGAATT |
Primer Names | Primer Sequences (5′-3′) |
---|---|
PRRSV N-F | ATAACAACGGCAAGCAGCAG |
PRRSV N-R | CTCTGGACTGGTTTTGTTG |
M-MyD88-F | TCTCTCCAGGTGCCCATCAGAAG |
M-MyD88-R | GCAAGGCGAGTCCAGAACCAAG |
M-TRIF-F | AGTCGCCACTGCAACTTTCTGTAG |
M-TRIF-R | GGCTGATGAAGGAGGAGGAGGAG |
M-MAVS-F | CCCGAAAGTGCCTGCCAACC |
M-MAVS-R | TTGGATGGTGCTGGATTGGTGAAC |
M-RIPK2-F | CCCTGCCAGTTCCTCAAGACAATG |
M-RIPK2-R | AGCCCTTTGAGACGCAGAAATGG |
M-ASC-F | GATGCCATCGACCTCACTGACAAG |
M-ASC-R | TCCGCCACCTCCTTCATGCC |
M-CARD9-F | ACTTCCACTTCCGTCTGCCTCTC |
M-CARD9-R | CCACAGTCCCGCCAGTCCTC |
M-BCL10-F | CCAGATGGAGCCACGAACAACC |
M-BCL10-R | GGATGGTACATGACAGTGGATGCC |
M-MALT1-F | CGAGGTTTGGGAAGGAAGACTTGC |
M-MALT1-R | GTGATAATGCCCTGCTGCTCCTG |
M-STING-F | TCGCTGTCCCAAGAGGTTCTCC |
M-STING-R | GGGCACCGCTGAGTTCTTCAAG |
M-β-actin-F | AGAAGATGACCCAGATCATGTTTGA |
M-β-actin-R | TCCATCACGATGCCAGTGGTA |
M-IFN-β-F | ATGGAGAAAGGACATTCTAACTGCAA |
M-IFN-β-R | AGAAGCACAACAGGAGAGCAATTT |
M-ISG15-F | CTCTGAGCATCCTGGTGAGGAA |
M-ISG15-R | CGAAGGTCAGCCAGAACAGGT |
M-IFIT1-F | TCAGGTCAAGGATAGTCTGGAGCA |
M-IFIT1-R | TGTCTAGGAATTCAATCTGGTCCAA |
M-TNF-α-F | CATGTTGTAGCAAACCCTCAAGC |
M-TNF-α-R | ATGGCACCACCAGCTGGTTAT |
S-IFN-β-F | TGAGCATTCTGCAGTACCTGA |
S-IFN-β-R | CCGGAGGTAATCTGTAAGTCTGT |
S-ISG56-F | ATGGGAGTTGGTCATTCAAGA |
S-ISG56-R | CAGGTGTTTCACATAGGCCA |
S-IL-8-F | CTGCAGTTCTGGCAAGAGTAAGT |
S-IL-8-R | CACTCTCAATCACTCTCAGTTCCT |
S-TNF-α-F | ATCGCCGTCTCCTACCAGA |
S-TNF-α-R | TCGATCATCCTTCTCCAGCT |
S-β-actin-F | ATGAAGATCAAGATCATCGCG |
S-β-actin-R | TCGTACTCCTGCTTGCTGATC |
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Xu, Y.; Ye, M.; Zhang, Y.; Sun, S.; Luo, J.; Jiang, S.; Zhang, J.; Liu, X.; Shao, Q.; Cao, Q.; et al. Screening of Porcine Innate Immune Adaptor Signaling Revealed Several Anti-PRRSV Signaling Pathways. Vaccines 2021, 9, 1176. https://doi.org/10.3390/vaccines9101176
Xu Y, Ye M, Zhang Y, Sun S, Luo J, Jiang S, Zhang J, Liu X, Shao Q, Cao Q, et al. Screening of Porcine Innate Immune Adaptor Signaling Revealed Several Anti-PRRSV Signaling Pathways. Vaccines. 2021; 9(10):1176. https://doi.org/10.3390/vaccines9101176
Chicago/Turabian StyleXu, Yulin, Mengxue Ye, Youwen Zhang, Shaohua Sun, Jia Luo, Sen Jiang, Jiajia Zhang, Xueliang Liu, Qi Shao, Qi Cao, and et al. 2021. "Screening of Porcine Innate Immune Adaptor Signaling Revealed Several Anti-PRRSV Signaling Pathways" Vaccines 9, no. 10: 1176. https://doi.org/10.3390/vaccines9101176