ESCRT Protein VPS4A Is Required for the Formation of Replication Centers and Replication of Human Coronavirus 229E (HCoV-229E)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. 229E Virus Infections, Growth Curves, and Virus Titrations
2.3. TCID50 Titer Assays
2.4. Lentivirus Production and Cell Selection
2.5. RNA Isolation and RT-qPCR
2.6. Antibodies and Reagents
2.7. Immunofluorescence Staining and Imaging
2.8. Transmission Electron Microscopy
2.9. smFISH of Viral RNA, Imaging, and Analysis
2.10. Statistical Analysis
3. Results
3.1. Functional VPS4A Was Required for the Formation of HCoV-229E Replication Centers
3.2. HCoV-229E Infection Triggers an Inflammatory Response in the Absence of a Functional VPS4A
3.3. Effect of Other ESCRT Proteins on 229E Virus Growth
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Name | Forward Primer | Reverse Primer |
---|---|---|
229E M 1 | 5′ CATACTATCAACCCATTCAACAAG 3′ | 5′ CACGGCAACTGTCATGTATT 3′ |
IL-6 | 5′ CTGCGCAGCTTTAAGGAGTT 3′ | 5′ TAAGTTCTGTGCCCAGTGGA 3′ |
ISG15 | 5′ GCTCCATGTCGGTGTCAGAG-3′ | 5′-CTCGAAGGTCAGCCAGAACAG 3′ |
MX1 | 5′-CTGTGCAGCCAGTATGAGGAG 3′ | 5′ CAGGGTGATTAGCTCATGACTG 3′ |
GAPDH | 5′ ACCCACTCCTCCACCTTTGAC 3′ | 5′ CTGTTGCTGTAGCCAAATTCGT 3′ |
IFN-β | 5′ CTCTCCTGTTGTGCTTCTCCAC 3′ | 5′ TAGTCTCATTCCAGCCAGTGCT 3′ |
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Kumar, R.; Maldonado, R.K.; Christensen, N.D.; Bewley, M.C.; Flanagan, J.M.; Buchkovich, N.J.; Parent, L.J. ESCRT Protein VPS4A Is Required for the Formation of Replication Centers and Replication of Human Coronavirus 229E (HCoV-229E). COVID 2024, 4, 1338-1354. https://doi.org/10.3390/covid4090095
Kumar R, Maldonado RK, Christensen ND, Bewley MC, Flanagan JM, Buchkovich NJ, Parent LJ. ESCRT Protein VPS4A Is Required for the Formation of Replication Centers and Replication of Human Coronavirus 229E (HCoV-229E). COVID. 2024; 4(9):1338-1354. https://doi.org/10.3390/covid4090095
Chicago/Turabian StyleKumar, Rinki, Rebecca Kaddis Maldonado, Neil D. Christensen, Maria C. Bewley, John M. Flanagan, Nicholas J. Buchkovich, and Leslie J. Parent. 2024. "ESCRT Protein VPS4A Is Required for the Formation of Replication Centers and Replication of Human Coronavirus 229E (HCoV-229E)" COVID 4, no. 9: 1338-1354. https://doi.org/10.3390/covid4090095