A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Reagents
2.2. Mice
2.3. In Silico Modeling
2.4. Plasmids and Site-Directed Mutagenesis
2.5. Transfections
2.6. Western Immunoblots
2.7. Reverse Genetics
2.8. Virus Infection
2.8.1. In Vitro
2.8.2. In Vivo
2.8.3. IFN-β Treatment In Vivo
2.9. Plaque Assay
2.10. RNA Extraction and cDNA Synthesis
2.11. qPCR
2.12. IFN-β ELISA
2.13. FACS Analysis of IFNAR1 and IFNAR2 Expression
2.14. Histology and Identification of Lung Neutrophils
2.15. Statistical Analyses
3. Results
3.1. A Y84F Mutation in the H5N1 NS1 Conserved Putative SH2-Binding Domain Affects the Ability for NS1 to Upregulate AKT Phosphorylation
3.2. A Y84F Mutation Abrogates NS1-Mediated Inhibition of Type I IFN Signaling
3.3. Effects of the Conserved Putative SH2-Binding Domain in NS1 on Virus Replication
3.4. The Conserved Putative SH2-Binding Domain in NS1 Contributes to IAV Virulence In Vivo, Affecting the IFN Response
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Gene | Forward Primer (5′-3′) | Reverse Primer (5′-3′) |
---|---|---|
(h) HPRT1 | TCCTCCTCTGCTCCGCCACC | TCACTAATCACGACGCCAGGGCT |
(h) MxA | GATGATCAAAGGGATGTGGC | AGCTCGGCAACAGACTCTTC |
(h) EIF2AK2 | ACTTGGCCAAATCCACCTG | CCCAGATTTGACCTTCCTGA |
(m) HPRT1 | CATAACCTGGTTCATCATCGC | TCCTCCTCAGACCGCTTTT |
(m) ISG15 | CCCCAGCATCTTCACCTTTA | TGACTGTGAGAGCAAGCAGC |
(m) OAS1 | AGTTCTCCTCCACCTGCTCA | GGCTGTGGTACCCATGTTTT |
(m) EIF2AK2 | CTGTTGCAAGGCCAAAGTCT | GAACAAATCGTGACCGGAGT |
(m) IFNA4 | TATGTCCTCACAGCCAGCAG | TTCTGCAATGACCTCCATCA |
(m) IFNB1 | CCCAGTGCTGGAGAAATTGT | CCCTATGGAGATGACGGAGA |
(m) CXCL1 | TCTCCGTTCCTTGGGGACAC | CCACACTCAAGAATGGTCGC |
(m) CXCL2 | TCCAGGTCAGTTAGCCTTGC | CGGTCAAAAAGTTTGCCTTG |
(IAV) M | AGATGAGTCTTCTAACCGAGGTCG | TGCAAAAACATCTTCAAGTCTCTG |
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Wang, B.X.; Wei, L.; Kotra, L.P.; Brown, E.G.; Fish, E.N. A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection. Viruses 2017, 9, 107. https://doi.org/10.3390/v9050107
Wang BX, Wei L, Kotra LP, Brown EG, Fish EN. A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection. Viruses. 2017; 9(5):107. https://doi.org/10.3390/v9050107
Chicago/Turabian StyleWang, Ben X., Lianhu Wei, Lakshmi P. Kotra, Earl G. Brown, and Eleanor N. Fish. 2017. "A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection" Viruses 9, no. 5: 107. https://doi.org/10.3390/v9050107
APA StyleWang, B. X., Wei, L., Kotra, L. P., Brown, E. G., & Fish, E. N. (2017). A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection. Viruses, 9(5), 107. https://doi.org/10.3390/v9050107