Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1.

    Kash, J. C. & Taubenberger, J. K. The role of viral, host, and secondary bacterial factors in influenza pathogenesis. Am. J. Pathol. 185, 1528–1536 (2015).

    Article  Google Scholar 

  2. 2.

    Kash, J. C. et al. Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature 443, 578–581 (2006).

    CAS  Article  Google Scholar 

  3. 3.

    Kobasa, D. et al. Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature 445, 319–323 (2007).

    CAS  Article  Google Scholar 

  4. 4.

    de Jong, M. D. et al. Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat. Med. 12, 1203–1207 (2006).

    Article  Google Scholar 

  5. 5.

    Pichlmair, A. et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science 314, 997–1001 (2006).

    CAS  Article  Google Scholar 

  6. 6.

    te Velthuis, A. J. W. & Fodor, E. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis. Nat. Rev. Microbiol. 14, 479–493 (2016).

    CAS  Article  Google Scholar 

  7. 7.

    Kowalinski, E. et al. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell 147, 423–435 (2011).

    CAS  Article  Google Scholar 

  8. 8.

    Lee, M.-K. et al. Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5′-triphosphate. Nucleic Acids Res. 44, 8407–8416 (2016).

    CAS  Article  Google Scholar 

  9. 9.

    Robb, N. C. et al. Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA. Nucleic Acids Res. 44, 10304–10315 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Russell, A. B., Trapnell, C. & Bloom, J. D. Extreme heterogeneity of influenza virus infection in single cells. eLife 7, e32303 (2018).

    Article  Google Scholar 

  11. 11.

    Killip, M. J., Jackson, D., Pérez-Cidoncha, M., Fodor, E. & Randall, R. E. Single-cell studies of IFN-β promoter activation by wild-type and NS1-defective influenza A viruses. J. Gen. Virol. 98, 357–363 (2017).

    CAS  Article  Google Scholar 

  12. 12.

    Killip, M. J., Fodor, E. & Randall, R. E. Influenza virus activation of the interferon system. Virus Res. 209, 11–22 (2015).

    CAS  Article  Google Scholar 

  13. 13.

    Jennings, P. A., Finch, J. T., Winter, G. & Robertson, J. S. Does the higher order structure of the influenza virus ribonucleoprotein guide sequence rearrangements in influenza viral RNA? Cell 34, 619–627 (1983).

    CAS  Article  Google Scholar 

  14. 14.

    Perez, J. T. et al. Influenza A virus-generated small RNAs regulate the switch from transcription to replication. Proc. Natl Acad. Sci. USA 107, 11525–11530 (2010).

    CAS  Article  Google Scholar 

  15. 15.

    Coloma, R. et al. The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog. 5, e1000491 (2009).

    Article  Google Scholar 

  16. 16.

    Li, H. et al. Internal genes of a highly pathogenic H5N1 influenza virus determine high viral replication in myeloid cells and severe outcome of infection in mice. PLoS Pathog. 14, e1006821 (2018).

    Article  Google Scholar 

  17. 17.

    Turrell, L., Lyall, J. W., Tiley, L. S., Fodor, E. & Vreede, F. T. The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes. Nat. Commun. 4, 1591 (2013).

    Article  Google Scholar 

  18. 18.

    Kato, H. et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441, 101–105 (2006).

    CAS  Article  Google Scholar 

  19. 19.

    Rehwinkel, J. et al. RIG-I detects viral genomic RNA during negative-strand RNA virus infection. Cell 140, 397–408 (2010).

    CAS  Article  Google Scholar 

  20. 20.

    Killip, M. J., Smith, M., Jackson, D. & Randall, R. E. Activation of the interferon induction cascade by influenza A viruses requires viral RNA synthesis and nuclear export. J. Virol. 88, 3942–3952 (2014).

    Article  Google Scholar 

  21. 21.

    Dulin, D. et al. Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site. Nucleic Acids Res. 43, 10421–10429 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Cheung, P. P. H. et al. Generation and characterization of influenza A viruses with altered polymerase fidelity. Nat. Commun. 5, 4794 (2014).

    CAS  Article  Google Scholar 

  23. 23.

    Woodman, A., Arnold, J. J., Cameron, C. E. & Evans, D. J. Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination. Nucleic Acids Res. 44, 6883–6895 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Forero, A. et al. The 1918 influenza virus PB2 protein enhances virulence through the disruption of inflammatory and Wnt-mediated signaling in mice. J. Virol. 90, 2240–2253 (2016).

    CAS  Article  Google Scholar 

  25. 25.

    Miotto, O., Heiny, A. T., Tan, T., August, J. T. & Brusic, V. Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by large-scale mutual information analysis. BMC Bioinformatics 9, S18 (2008).

    Article  Google Scholar 

  26. 26.

    Graef, K. M. et al. The PB2 subunit of the influenza virus RNA polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon. J. Virol. 84, 8433–8445 (2010).

    CAS  Article  Google Scholar 

  27. 27.

    Du, Y. et al. Genome-wide identification of interferon-sensitive mutations enables influenza vaccine design. Science 359, 290–296 (2018).

    CAS  Article  Google Scholar 

  28. 28.

    Chan, F. K.-M., Luz, N. F. & Moriwaki, K. Programmed necrosis in the cross talk of cell death and inflammation. Annu. Rev. Immunol. 33, 79–106 (2015).

    CAS  Article  Google Scholar 

  29. 29.

    van den Brand, J. M. A. et al. Comparison of temporal and spatial dynamics of seasonal H3N2, pandemic H1N1 and highly pathogenic avian influenza H5N1 virus infections in ferrets. PLoS ONE 7, e42343 (2012).

    Article  Google Scholar 

  30. 30.

    de Wit, E. et al. 1918 H1N1 influenza virus replicates and induces pro-inflammatory cytokine responses in extra-respiratory tissues of ferrets. J. Infect. Dis. 217, 1237–1246 (2018).

    Article  Google Scholar 

  31. 31.

    Fodor, E. et al. A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J. Virol. 76, 8989–9001 (2002).

    CAS  Article  Google Scholar 

  32. 32.

    Kashiwagi, T., Leung, B. W., Deng, T., Chen, H. & Brownlee, G. G. The N-terminal region of the PA subunit of the RNA polymerase of influenza A/HongKong/156/97 (H5N1) influences promoter binding. PLoS ONE 4, e5473 (2009).

    Article  Google Scholar 

  33. 33.

    Tumpey, T. M. et al. Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 310, 77–80 (2005).

    CAS  Article  Google Scholar 

  34. 34.

    Vreede, F. T., Jung, T. E. & Brownlee, G. G. Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates. J. Virol. 78, 9568–9572 (2004).

    CAS  Article  Google Scholar 

  35. 35.

    Fodor, E. et al. Rescue of influenza A virus from recombinant DNA. J. Virol. 73, 9679–9682 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Childs, K. S., Andrejeva, J., Randall, R. E. & Goodbourn, S. Mechanism of mda-5 inhibition by paramyxovirus V proteins. J. Virol. 83, 1465–1473 (2009).

    CAS  Article  Google Scholar 

  37. 37.

    Zettl, M., Adrain, C., Strisovsky, K., Lastun, V. & Freeman, M. Rhomboid family pseudoproteases use the ER quality control machinery to regulate intercellular signaling. Cell 145, 79–91 (2011).

    CAS  Article  Google Scholar 

  38. 38.

    Hertzog, J. et al. Infection with a Brazilian isolate of Zika virus generates RIG-I stimulatory RNA and the viral NS5 protein blocks type I IFN induction and signaling. Eur. J. Immunol. 48, 1120–1136 (2018).

    CAS  Article  Google Scholar 

  39. 39.

    Oymans, J. & te Velthuis, A. J. W. A mechanism for prime-realignment during influenza A virus replication. J. Virol. 92, e01773-17 (2018).

    Article  Google Scholar 

  40. 40.

    Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).

    CAS  Article  Google Scholar 

  41. 41.

    Li, H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27, 2987–2993 (2011).

    CAS  Article  Google Scholar 

  42. 42.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2012).

    Article  Google Scholar 

  43. 43.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.J. 17, 10 (2011).

    Article  Google Scholar 

  44. 44.

    Smith, T., Heger, A. & Sudbery, I. UMI-tools: modeling sequencing errors in unique molecular identifiers to improve quantification accuracy. Genome Res. 27, 491–499 (2017).

    CAS  Article  Google Scholar 

  45. 45.

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  46. 46.

    Leung, Y. Y. et al. DASHR: database of small human noncoding RNAs. Nucleic Acids Res. 44, D216–D222 (2016).

    CAS  Article  Google Scholar 

  47. 47.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  Google Scholar 

  48. 48.

    Ignatiadis, N., Klaus, B., Zaugg, J. B. & Huber, W. Data-driven hypothesis weighting increases detection power in genome-scale multiple testing. Nat. Methods 13, 577–580 (2016).

    CAS  Article  Google Scholar 

  49. 49.

    Kim, S.-Y. & Volsky, D. J. PAGE: parametric analysis of gene set enrichment. BMC Bioinformatics 6, 144 (2005).

    Article  Google Scholar 

  50. 50.

    Durinck, S., Spellman, P. T., Birney, E. & Huber, W. Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat. Protoc. 4, 1184–1191 (2009).

    CAS  Article  Google Scholar 

Download references


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.

Author information




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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing