Mini viral RNAs act as innate immune agonists during influenza virus infection
te Velthuis A., Long J., Bauer DLV., Fan R., Yen H-L., Sharps J., Siegers J., Killip M., French H., Oliva-Martin MJ., Randall R., de Wit E., van Riel D., Poon L., Fodor E.
Influenza A virus infection usually causes a mild to moderately severe respiratory disease in humans. However, infection with the 1918 H1N1 pandemic or highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype, can lead to viral pneumonia, systemic disease and death. The molecular processes that determine the outcome of influenza virus infection are multifactorial and involve a complex interplay between host, viral, and bacterial factors. However, it is generally accepted that a strong innate immune dysregulation known as 'cytokine storm' contributes to the pathology of pandemic and avian influenza virus infections. 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 (IFN) expression. 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 and activate the intracellular pathogen sensor RIG-I, and lead to the expression of interferon-beta. 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 and propose an intramolecular copy-choice mechanism for mvRNA generation. By deep-sequencing RNA samples from lungs of ferrets infected with influenza viruses we show that mvRNAs are generated during infection of animal models. We propose that mvRNAs act as main agonists of RIG-I during influenza virus infection and the ability of influenza virus strains to generate mvRNAs should be considered when assessing their virulence potential.