Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication
Abstract
:1. Introduction
2. Results
2.1. Identification and Sequence Analysis of EcTRAF4
2.2. Tissue Expression Analysis of EcTRAF4
2.3. Intracellular Localization of EcTRAF4
2.4. EcTRAF4 Overexpression Promotes RGNNV Replication
2.5. EcTRAF4 Knockdown Inhibits RGNNV Replication
2.6. RGNNV CP Interacts with EcTRAF4
2.7. RGNNV or CP Promotes EcTRAF4 Expression
2.8. EcTRAF4 Is a Negative Regulator of Virus-Induced IFN Signaling
3. Discussion
4. Materials and Methods
4.1. Fish, Cell Lines, and Virus
4.2. RNA Isolation, cDNA Synthesis, and RT–qPCR
4.3. Cloning of EcTRAF4 and Sequence Analysis
4.4. Plasmid Construction
4.5. Cell Transfection
4.6. Cellular Localization Analysis
4.7. Viral Infection Assays and Sample Collection
4.8. Small Interfering RNA (siRNA)-Mediated EcTRAF4 Knockdown
4.9. Coimmunoprecipitation Assays and Western Blotting
4.10. Yeast Two-Hybrid Analysis
4.11. RT–qPCR Analysis of Relative Expression Levels of host and Viral Genes
4.12. Dual-Luciferase Reporter Assays
4.13. Statistical Analyses
4.14. Ethics Statement
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Name | Sequence (5′–3′) |
---|---|
RGNNV-CP-RT-F | CAACTGACAACGATCACACCTTC |
RGNNV-CP-RT-R | CAATCGAACACTCCAGCGACA |
RGNNV-RdRp-RT-F | GTGTCCGGAGAGGTTAAGGATG |
RGNNV-RdRp-RT-R | CTTGAATTGATCAACGGTGAACA |
EcTRAF4-RT-F | TTCAGCCCACCCTTCTACACTCA |
EcTRAF4-RT-R | CCACTCCAGCAGGTTGTCGTACTCT |
β-actin-RT-F β-actin-RT-R | TACGAGCTGCCTGACGGACA GGCTGTGATCTCCTTCTGCA |
EcISG15-RT-F | CCTATGACATCAAAGCTGACGAGAC |
EcISG15-RT-R | GTGCTGTTGGCAGTGACGTTGTAGT |
EcISG56-RT-F | CTGTTGTTACGCACGGAGGAT |
EcISG56-RT-R | CCTGCGTGGGTTCATTCAGT |
EcIFN2-RT-S | TACAGCCAGGCGTCCAAAGCATC |
EcIFN2-RT-R | CAGTACAGGAGCGAAGGCCGACA |
EcIL-1β-RT-F | AACCTCATCATCGCCACACA |
EcIL-1β-RT-R | AGTTGCCTCACAACCGAACAC |
EcIL8-RT-F | GCCGTCAGTGAAGGGAGTCTAG |
EcIL8-RT-R | ATCGCAGTGGGAGTTTGCA |
EcTNFα-RT-F | GTGTCCTGCTGTTTGCTTGGTA |
EcTNFα-RT-R | CAGTGTCCGACTTGATTAGTGCTT |
EcTRAF6-RT-F | CCCTATCTGCCTTATGGCTTTGA |
EcTRAF6-RT-R | ACAGCGGACAGTTAGCGAGAGTAT |
Name | Sequence (5′–3′) | Usage |
---|---|---|
F1 | ATGCCCGGGTTTGATTACAAGTTTC | EcTRAF4 cloning |
R1 | TTAAGCCATGATCTTCTGGGGAATC | |
C1-EcTRAF4-F C1-EcTRAF4-R | GCCTCGAGCTATGCCCGGGTTTGATTAC GCGGATCCTTAAGCCATGATCTTCTGG | pEGFP-C1 cloning |
HA-EcTRAF4-F | GCGGATCCTATGCCCGGGTTTGATTAC | pcDNA3.1-3HA cloning |
HA-EcTRAF4-R | GCCTCGAGTTAAGCCATGATCTTCTGG | |
HA-CP-F | GCAAGCTTATGGTACGCAAAGGTGAGAAGAAAT | pcDNA3.1-3HA cloning |
HA-CP-R | GCGAATTCGTTTTCCGAGTCAACCCTGGTGCAG |
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Wu, S.; Sun, M.; Zhang, X.; Liao, J.; Liu, M.; Qin, Q.; Wei, J. Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication. Int. J. Mol. Sci. 2021, 22, 6136. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22116136
Wu S, Sun M, Zhang X, Liao J, Liu M, Qin Q, Wei J. Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication. International Journal of Molecular Sciences. 2021; 22(11):6136. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22116136
Chicago/Turabian StyleWu, Siting, Mengshi Sun, Xin Zhang, Jiaming Liao, Mengke Liu, Qiwei Qin, and Jingguang Wei. 2021. "Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication" International Journal of Molecular Sciences 22, no. 11: 6136. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22116136