Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

Open Access
Publisher Website
Google Scholar
Abstract
Activation of the latent kinase PKR is a potent innate defense reaction of vertebrate cells towards viral infections, which is triggered by recognition of viral double-stranded (ds) RNA and results in a translational shutdown. A major gap in our understanding of PKR's antiviral properties concerns the nature of the kinase activating molecules expressed by influenza and other viruses with a negative strand RNA genome, as these pathogens produce little or no detectable amounts of dsRNA. Here we systematically investigated PKR activation by influenza B virus and its impact on viral pathogenicity. Biochemical analysis revealed that PKR is activated by viral ribonucleoprotein (vRNP) complexes known to contain single-stranded RNA with a 5′-triphosphate group. Cell biological examination of recombinant viruses showed that the nucleo-cytoplasmic transport of vRNP late in infection is a strong trigger for PKR activation. In addition, our analysis provides a mechanistic explanation for the previously observed suppression of PKR activation by the influenza B virus NS1 protein, which we show here to rely on complex formation between PKR and NS1's dsRNA binding domain. The high significance of this interaction for pathogenicity was revealed by the finding that attenuated influenza viruses expressing dsRNA binding-deficient NS1 proteins were rescued for high replication and virulence in PKR-deficient cells and mice, respectively. Collectively, our study provides new insights into an important antiviral defense mechanism of vertebrates and leads us to suggest a new model of PKR activation by cytosolic vRNP complexes, a model that may also be applicable to other negative strand RNA viruses. Upon viral infection of vertebrate cells, a vigorous innate defense response is initiated via the recognition of viral double-stranded (ds) RNA by the protein kinase PKR, resulting in the cessation of protein synthesis and subsequent blockage of viral propagation. The activation of PKR's potent antiviral response against influenza and other viruses with a negative strand RNA genome has presented a conundrum, however, as previous attempts failed to detect dsRNA in cells infected with these viruses. Here, we identify genomic RNA within the ribonucleoprotein (RNP) of influenza viruses as a non-canonical activator of the latent kinase PKR. Cell biological examinations revealed that the transfer of viral RNP from the nucleus to the cytoplasm provides a strong stimulus for PKR activation. Moreover, we provide insight into mechanisms of pathogenesis by showing PKR and the NS1 protein of influenza B virus forms a complex in infected cells, which inhibits PKR activation. This interaction seems to be crucial for viral pathogenicity, as a strong attenuation of NS1 mutant viruses was largely rescued in PKR-deficient mice and cells. Taken together, these findings suggest a new model for the induction and inhibition of PKR by influenza virus that may also apply to viruses with a similar genome structure.