Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. Viruses and Infection Conditions
2.3. Poly(A)+ RNA-Selection and Isolation of gRNA and sgRNA
2.4. Quantitation of RNA Modifications by LC-MS/MS Analysis
2.5. Generation of In Vitro Transcribed (IVT) CHIKV RNA
2.6. IFN Pre-Treatment
2.7. siRNA Knockdown and ADAR1 Overexpression
2.8. Western Blotting
2.9. Two-Step Quantitative Reverse Transcription PCR (qRT-PCR)
2.10. Cell Viability Assay
2.11. Library Preparation for RNA-Seq
2.12. ADAR1 Editing Analyses
2.13. Statistical Analysis and Chart Production
3. Results
3.1. Mass Spectrometry Analyses Reveal Inosine Enrichment in the CHIKV Genomic RNA but Not in the Subgenomic One
3.2. CHIKV Infection Does Not Alter the Induction of ADAR1 Isoforms
3.3. ADAR1 Knockdown Inhibits CHIKV RNA and Protein Levels, and Virion Production
3.4. ADAR1 Knockdown Leads to Phosphorylation of Eukaryotic Translation Initiation Factor 2α (eIF2α)
3.5. RNA-Seq Analyses Detect Adenosine to Inosine (A-to-I) Editing in CHIKV RNAs
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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Baquero-Pérez, B.; Bortoletto, E.; Rosani, U.; Delgado-Tejedor, A.; Medina, R.; Novoa, E.M.; Venier, P.; Díez, J. Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus. Viruses 2024, 16, 945. https://doi.org/10.3390/v16060945
Baquero-Pérez B, Bortoletto E, Rosani U, Delgado-Tejedor A, Medina R, Novoa EM, Venier P, Díez J. Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus. Viruses. 2024; 16(6):945. https://doi.org/10.3390/v16060945
Chicago/Turabian StyleBaquero-Pérez, Belinda, Enrico Bortoletto, Umberto Rosani, Anna Delgado-Tejedor, Rebeca Medina, Eva Maria Novoa, Paola Venier, and Juana Díez. 2024. "Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus" Viruses 16, no. 6: 945. https://doi.org/10.3390/v16060945