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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

1
Department of Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany
2
Department of Computational Molecular Biology, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
3
Sequencing Core Facility, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
4
Bioprocess Engineering, Otto von Guericke University Magdeburg, 39106 Magdeburg, Germany
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Viruses 2020, 12(1), 71; https://doi.org/10.3390/v12010071
Received: 28 November 2019 / Revised: 20 December 2019 / Accepted: 3 January 2020 / Published: 7 January 2020
(This article belongs to the Section Animal Viruses)
Virus replication displays a large cell-to-cell heterogeneity; yet, not all sources of this variability are known. Here, we study the effect of defective interfering (DI) particle (DIP) co-infection on cell-to-cell variability in influenza A virus (IAV) replication. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNA) and are, thus, defective in virus replication. Moreover, they interfere with virus replication. Using single-cell isolation and reverse transcription polymerase chain reaction, we uncovered a large between-cell heterogeneity in the DI vRNA content of infected cells, which was confirmed for DI mRNAs by single-cell RNA sequencing. A high load of intracellular DI vRNAs and DI mRNAs was found in low-productive cells, indicating their contribution to the large cell-to-cell variability in virus release. Furthermore, we show that the magnitude of host cell mRNA expression (some factors may inhibit virus replication), but not the ribosome content, may further affect the strength of single-cell virus replication. Finally, we show that the load of viral mRNAs (facilitating viral protein production) and the DI mRNA content are, independently from one another, connected with single-cell virus production. Together, these insights advance single-cell virology research toward the elucidation of the complex multi-parametric origin of the large cell-to-cell heterogeneity in virus infections. View Full-Text
Keywords: single-cell analysis; influenza A virus; cell-to-cell heterogeneity; defective interfering particles; single-cell RNA sequencing; next-generation sequencing single-cell analysis; influenza A virus; cell-to-cell heterogeneity; defective interfering particles; single-cell RNA sequencing; next-generation sequencing
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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.

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