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Mini viral RNAs act as innate immune agonists during influenza virus infection

Abstract

The molecular processes that determine the outcome of influenza virus infection in humans are multifactorial and involve a complex interplay between host, viral and bacterial factors1. However, it is generally accepted that a strong innate immune dysregulation known as ‘cytokine storm’ contributes to the pathology of infections with the 1918 H1N1 pandemic or the highly pathogenic avian influenza viruses of the H5N1 subtype2,3,4. The RNA sensor retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral infection and initiating a signalling cascade that leads to interferon expression5. Here, we show that short aberrant RNAs (mini viral RNAs (mvRNAs)), produced by the viral RNA polymerase during the replication of the viral RNA genome, bind to and activate RIG-I and lead to the expression of interferon-β. We find that erroneous polymerase activity, dysregulation of viral RNA replication or the presence of avian-specific amino acids underlie mvRNA generation and cytokine expression in mammalian cells. By deep sequencing RNA samples from the lungs of ferrets infected with influenza viruses, we show that mvRNAs are generated during infection in vivo. We propose that mvRNAs act as the main agonists of RIG-I during influenza virus infection.

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Fig. 1: mvRNAs of influenza A virus are bound by RIG-I and induce IFN expression.
Fig. 2: Dysregulation of RNA replication in cells infected with WSN results in the generation of mvRNAs.
Fig. 3: The PB2 polymerase subunit of highly virulent influenza A viruses promotes mvRNA synthesis.
Fig. 4: Levels of mvRNAs produced during infection correlate with innate immune responses.

Data availability

All sequencing data have been deposited in the NCBI Sequence Read Archive under accession number SRP158565. Gene expression data are available as Supplementary Data 1. Supplementary figures and tables are available in the Supplementary Information file.

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Acknowledgements

We greatly value our discussions with R. Sun and Y. Du, who independently found that mutations in the N-terminal region of PB2, near the template exit channel of the polymerase, stimulate IFN induction27. We thank G. G. Brownlee, M. Freeman and F. Vreede for plasmids, J. Rehwinkel and A. Mayer (all from University of Oxford) for HEK293T RIG-I−/− cells, Y. Kawaoka (University of Wisconsin–Madison) for the A/Brevig Mission/1/1918 (H1N1) virus, and A. Osterhaus and T. Kuiken (both from Erasmus Medical Centre) for the ferret tissue samples infected with A/Indonesia/5/2005 (H5N1) and A/Netherlands/602/2009 (H1N1). We thank I. Sudbery (University of Sheffield) for adding spliced read functionality to the umi_tools package. We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (funded by Wellcome Trust grant 090532/Z/09/Z) for the generation of adapter-ligated mvRNA sequencing data. This work was supported by the Wellcome Trust grant 098721/Z/12/Z, the joint Wellcome Trust and Royal Society grant 206579/Z/17/Z and a Netherlands Organization for Scientific Research (NWO) grant 825.11.029 to A.J.W.t.V.; EPA Cephalosporin Junior Research Fellowship to D.L.V.B.; support by the Intramural Research Program of NIAID, NIH, to E.d.W.; Research Grants Council of the Hong Kong Special Administrative Region, China, project no. T11-705/14N and a Croucher Senior Research Fellowship to L.L.M.P.; and Medical Research Council (MRC) programme grants MR/K000241/1 and MR/R009945/1 to E.F. and studentship to J.C.L.

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Contributions

J.C.L., E.F., J.S. and A.J.W.t.V. showed that subgenomic influenza vRNAs stimulate IFN-β production and are bound by RIG-I. A.J.W.t.V. and J.C.L. found that mvRNAs are produced by influenza virus polymerases. D.L.V.B. designed the sequencing strategies. D.L.V.B. and A.J.W.t.V. performed the deep-sequencing experiments and analyses. M.J.K., M.J.O.-M., H.F. and R.E.R. contributed reagents and protocols. E.d.W., D.v.R. and J.Y.S. provided the ferret lung tissues. R.L.Y.F., H.-L.Y. and L.L.M.P. performed the A549 infections. A.J.W.t.V., D.L.V.B., J.C.L. and E.F. analysed the data. A.J.W.t.V., J.C.L., D.L.V.B. and E.F. wrote the manuscript with input from co-authors.

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Correspondence to Aartjan J. W. te Velthuis or Ervin Fodor.

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Supplementary information

Supplementary Information

Supplementary Figures 1–5, Supplementary Tables 1–3, Raw Images for Figs 1–4, Raw Images for Supplementary Figures 1, 3 and 4.

Reporting Summary

Supplementary Table 1

Gene expression in response to mvRNA levels.

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te Velthuis, A.J.W., Long, J.C., Bauer, D.L.V. et al. Mini viral RNAs act as innate immune agonists during influenza virus infection. Nat Microbiol 3, 1234–1242 (2018). https://doi.org/10.1038/s41564-018-0240-5

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