Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Viruses
2.2. Isolation of Infected Single Cells
2.3. Plaque Assay
2.4. Single-Cell reverse transcription polymerase chain reaction (RT-PCR)
2.5. Quantification of Ribosomes
2.6. Cell Population-Based Infection and Analysis
2.7. Procedure for Single-Cell RNA sequencing (scRNA-Seq)
2.8. Data Processing and Quality Control
- --
- outFilterMultimapNmax 5
- --
- outFilterScoreMinOverLread 0.25
- --
- outFilterMatchNminOverLread 0.25
- --
- outSJfilterOverhangMin 10 10 10 10
- --
- outSJfilterCountUniqueMin 1 1 1 1
- --
- outSJfilterCountTotalMin 1 1 1 1
2.9. Analysis of Deletion Junctions
2.10. Data and Software Availability
3. Results
3.1. Single-Cell Analysis of influenza A virus (IAV)-Infected Cells Demonstrates A Large Cell-to-Cell Heterogeneity in Intracellular Defective Interfering (DI) Viral RNA (vRNA) Content
3.2. Infected Single Cells with a Low Progeny Virus Titer Show a High Load of Intracellular DI vRNAs
3.3. scRNA-Seq Reveals a Decrease of Host Cell mRNA Fraction and an Increase of Viral mRNA Fraction in High-Yield Cells
3.4. scRNA-Seq Analysis Reveals an Association of the DI mRNA Content and the Single-Cell Virus Yield
3.5. De Novo Generation of DI vRNAs Observed at the Single-Cell Level
3.6. Viral mRNA Level and DI mRNA Content Are Both Connected with the Single-Cell Virus Yield, Independently from One Another
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Reaction | Target | Primer Name | Sequence (5′ -> 3′) |
---|---|---|---|
RT | All segments | Uni12 | AGCAAAAGCAGG |
PCR | Segment 1 | S1 Uni for | AGCGAAAGCAGGTCAATTAT |
S1 Uni rev | AGTAGAAACAAGGTCGTTTTTAAAC | ||
Segment 2 | S2 Uni for | AGCGAAAGCAGGCAAACC | |
S2 Uni rev | AGTAGGAACAAGGCATTTTTTCATG | ||
Segment 3 | S3 Uni for | AGCGAAAGAAGGTACTGATCC | |
S3 Uni rev | AGTAGAAACAAGGTACTTTTTTGGAC |
Reaction | Target | Primer Name | Sequence (5′ -> 3′) |
---|---|---|---|
PCR | Segment 4 | S4 Uni for | AGCAAAAGCAGGGGAA |
S4 Uni rev | AGTAGAAACAAGGGTGTTTT | ||
Segment 5 | S5 Uni for | AGCAAAAGCAGGGTAGATAATC | |
S5 Uni rev | AGTAGAAACAAGGGTATTTTTC | ||
Segment 6 | S6 Uni for | AGCGAAAGCAGGGGTTTAAAATG | |
S6 Uni rev | AGTAGAAACAAGGAGTTTTTTGAAC | ||
Segment 7 | S7 Uni for | AGCGAAAGCAGGTAGATATTG | |
S7 Uni rev | AGTAGAAACAAGGTAGTTTTTTAC | ||
Segment 8 | S8 Uni for | AGAAAAAGCAGGGTGACAAA | |
S8 Uni rev | AGTAGAAACAAGGGTGTTTT |
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Kupke, S.Y.; Ly, L.-H.; Börno, S.T.; Ruff, A.; Timmermann, B.; Vingron, M.; Haas, S.; Reichl, U. Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication. Viruses 2020, 12, 71. https://doi.org/10.3390/v12010071
Kupke SY, Ly L-H, Börno ST, Ruff A, Timmermann B, Vingron M, Haas S, Reichl U. Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication. Viruses. 2020; 12(1):71. https://doi.org/10.3390/v12010071
Chicago/Turabian StyleKupke, Sascha Young, Lam-Ha Ly, Stefan Thomas Börno, Alexander Ruff, Bernd Timmermann, Martin Vingron, Stefan Haas, and Udo Reichl. 2020. "Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication" Viruses 12, no. 1: 71. https://doi.org/10.3390/v12010071
APA StyleKupke, S. Y., Ly, L. -H., Börno, S. T., Ruff, A., Timmermann, B., Vingron, M., Haas, S., & Reichl, U. (2020). Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication. Viruses, 12(1), 71. https://doi.org/10.3390/v12010071