Asparagine Availability Is a Critical Limiting Factor for Infectious Spleen and Kidney Necrosis Virus Replication
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines and Viral Strains
2.2. Reagents and Antibodies
2.3. Cell Viability Assay
2.4. Glutamine Depletion and Rescue
2.5. Determination of TCID50 of ISKNV Virus
2.6. The Transcriptional Expression of Genes Was Detected by qPCR
2.7. DNA Extraction and Quantification of ISKNV Genome Copies
2.8. Western Blot
2.9. RNA Interference
2.10. Statistical Analysis
3. Results
3.1. ISKNV Infection Altered Asparagine Metabolism
3.2. Inhibition of the MAS-Affected ISKNV Replication
3.3. ASNS Knockdown Reduced ISKNV Replication
3.4. Asparagine Fully Rescued ISKNV Replication in Glutamine Depletion
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer Name | Gene Name | Sequence (5′-3′) |
---|---|---|
ASNS-F | asparagine synthetase | GAAATGTGTGGTATCTGGGCTTTGT |
ASNS-R | TCAAAGACTTCAAAGTGGCCAGGG | |
MDH2-F | malate dehydrogenase 2 | ACTGCCCCGAAGCTATGA |
MDH2-R | CAGCGTGACCTCCAATGAC | |
GOT2-F | Glutamic-oxaloacetic transaminase | ACCATGGCCCTGCTCAAG |
GOT2-R | TACTTGGTGACAGCATGGATCGC | |
GOT1-F | Glutamic-oxaloacetic transaminase 1 | CGGCTTATCAGGGTTTCGC |
GOT1-R | CAGGGCTGTTGAGGGTCTTG | |
MDH1-F | malate dehydrogenase 1 | ATGGGTGTTTACTCCTCTGGC |
MDH1-R | TTTGGGGCGGTCTAAGTG | |
18S-F | 18S RNA | CATTCGTATTGTGCCGCTAGA |
18S-R | CAAATGCTTTCGCTTTGGTC | |
ISKNV-F | ISKNV-007 | CGAGGCCACATCCAACATC |
SKNV-R | CGCCTTTAACGTGGGATATATTG | |
ISKNV-Probe | CACCAAACTGACCGCGGACTCGT |
Name | Sequence (5′-3′) | Anti-Sequence (5′-3′) |
---|---|---|
siRNA-ASNS (siASNS-1) | GGCUUUGUUUGGCAGUGAUTT | AUCACUGCCAAACAAAGCCTT |
siRNA-ASNS (siASNS-2) | GCAAGGUUGCAGCUCACAUTT | AUGUGAGCUGCAACCUUGCTT |
iRNA-ASNS (siASNS-3) | GGUAUGUACUUGGUUUCAATT | UUGAAACCAAGUACAUACCTT |
Negative control (NC) | UUCUCCGAACGUGUCACGUTT | ACGUGACACGUUCGGAGAATT |
Sample | Intracellular (TCID50/0.1 mL) | Supernatant (TCID50/0.1 mL) | Complete (TCID50/0.1 mL) | |
---|---|---|---|---|
AOOA Concentration | ||||
control | 10−4.23 | 10−4.16 | 10−4.71 | |
50 μM | 10−3.78 | 10−3.71 | 10−4.33 | |
100 μM | 10−3.29 | 10−3.13 | 10−3.6 |
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Ma, B.; Li, F.; Fu, X.; Luo, X.; Lin, Q.; Liang, H.; Niu, Y.; Li, N. Asparagine Availability Is a Critical Limiting Factor for Infectious Spleen and Kidney Necrosis Virus Replication. Viruses 2024, 16, 1540. https://doi.org/10.3390/v16101540
Ma B, Li F, Fu X, Luo X, Lin Q, Liang H, Niu Y, Li N. Asparagine Availability Is a Critical Limiting Factor for Infectious Spleen and Kidney Necrosis Virus Replication. Viruses. 2024; 16(10):1540. https://doi.org/10.3390/v16101540
Chicago/Turabian StyleMa, Baofu, Fangying Li, Xiaozhe Fu, Xia Luo, Qiang Lin, Hongru Liang, Yinjie Niu, and Ningqiu Li. 2024. "Asparagine Availability Is a Critical Limiting Factor for Infectious Spleen and Kidney Necrosis Virus Replication" Viruses 16, no. 10: 1540. https://doi.org/10.3390/v16101540
APA StyleMa, B., Li, F., Fu, X., Luo, X., Lin, Q., Liang, H., Niu, Y., & Li, N. (2024). Asparagine Availability Is a Critical Limiting Factor for Infectious Spleen and Kidney Necrosis Virus Replication. Viruses, 16(10), 1540. https://doi.org/10.3390/v16101540