Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

Boergeling, Yvonne; Rozhdestvensky, Timofey S.; Schmolke, Mirco; Resa-Infante, Patricia; Robeck, Thomas; Randau, Gerrit; Wolff, Thorsten; Gabriel, Gülşah; Brosius, Jürgen; Ludwig, Stephan

doi:10.1371/journal.ppat.1004924

Cited by 31 publications

(26 citation statements)

References 73 publications

(126 reference statements)

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“…A proposed hypothesis is that the unencapsidated replication products of small DVGs can potentially activate RIG-I if they can reach the cytoplasm [20], given the observations in vitro and in vivo of short influenza RNA template replication in the absence of nucleoprotein (NP) [29]. In addition, the interfering and immune-modulatory abilities of DVGs could also be attributed to DVG-encoded proteins that directly interact with mitochondrial antiviral-signaling (MAVS) and act independently of RIG-I in IFN induction, as previously reported [30].…”

Section: Discussionmentioning

confidence: 88%

Cell-to-cell variation in defective virus expression and effects on host responses during influenza virus infection

Wang

Forst

Chou

et al. 2018

Preprint

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16 17 ABSTRACT 18 Virus and host factors contribute to cell-to-cell variation in viral infections and determine the 19 outcome of the overall infection. However, the extent of the variability at the single cell level and 20 how it impacts virus-host interactions at a systems level are not well understood. To characterize 21 the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to 22 quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary 23 bronchial epithelial cells infected with influenza A virus. We observed substantial variability of viral 24 transcription between cells, including the accumulation of defective viral genomes (DVGs) that 25 impact viral replication. We show a correlation between DVGs and viral-induced variation of the 26 host transcriptional program and an association between differential induction of innate immune 27 response genes and attenuated viral transcription in subpopulations of cells. These observations 28 at the single cell level improve our understanding of the complex virus-host interplay during 29 influenza infection.30 31 IMPORTANCE 32 Defective influenza virus particles generated during viral replication carry incomplete viral 33 genomes and can interfere with the replication of competent viruses. These defective genomes 34 are thought to modulate disease severity and pathogenicity of the influenza infection. Different 35 defective viral genomes also introduce another source of variation across a heterogeneous cell 36 population. Evaluating the impact of defective virus genomes on host cell responses cannot be 37 fully resolved at the population level, requiring single cell transcriptional profiling. Here we 38 characterized virus and host transcriptomes in individual influenza-infected cells, including that of 39 defective viruses that arise during influenza A virus infection. We established an association 40 between defective virus transcription and host responses and validated interfering and 41 immunostimulatory functions of identified dominant defective virus genome species in vitro. This 42 study demonstrates the intricate effects of defective viral genomes on host transcriptional 43 responses and highlights the importance of capturing host-virus interactions at the single-cell level. 44 45 82 Cell-to-cell variation in virus gene expression. 83 To determine how both the viral and host cell transcriptional programs relate to each other over 84 the course of an influenza infection, we infected two cell types-the adenocarcinomic human 85 alveolar basal epithelial A549 cell line and human primary bronchial epithelial cells HBEpC-at 86 high multiplicity of Infection (MOI; 5) with A/Puerto Rico/8/34(H1N1) (PR8) and performed single 87 cell and bulk RNA-seq expression analyses. A high MOI infection ensures that virtually all the 88 cells can be rapidly infected, promotes the accumulation of DVGs, and consequently enables the 89 characterization of both host response and DVG diversity. We first determi...

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Section: Discussionmentioning

confidence: 88%

Cell-to-cell variation in defective virus expression and effects on host responses during influenza virus infection

Wang

Forst

Chou

et al. 2018

Preprint

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“…DI vRNAs typically retain the terminal 3 and 5 ends harboring the promoter regions, which are necessary for viral transcription [11,12]. Therefore, the majority, if not all, DI vRNAs are able to transcribe and generate DI mRNAs [54][55][56]. Thus, we continued analyzing DI mRNAs by scRNA-seq.…”

Section: Scrna-seq Analysis Reveals An Association Of the DI Mrna Conmentioning

confidence: 99%

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

Kupke

Börno

et al. 2020

Viruses

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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.

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“…4D). Recently, a truncated protein expressed from a DI of influenza A virus PB2 protein was implicated in the attenuation of the full-length virus in vitro (29). Because Dg and Dwt conserved an intact 5= end, they theoretically could be translated and yield a full nsP1 protein as well as a chimeric nsP2-E1 protein.…”

Section: Sinv Mutator Rdrp Presents Altered Replication Kinetics At Hmentioning

confidence: 99%

Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

Poirier

Mounce

Rozen-Gagnon

et al. 2016

J Virol

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Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden.IMPORTANCE Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains.

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Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

Cited by 31 publications

References 73 publications

Cell-to-cell variation in defective virus expression and effects on host responses during influenza virus infection

Cell-to-cell variation in defective virus expression and effects on host responses during influenza virus infection

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

Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

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