Abstract
Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Here we applied photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP–vRNA interactions in infected human cells. NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. Finally, we demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells.
Highlights
Influenza A virus nucleoprotein (NP) association with viral RNA is essential for packaging, but the pattern of NP binding to vRNA is unclear
We show that the NP of Influenza A virus (IAV) binds the vRNA non-uniformly and that regions of low-NP binding are enriched for predicted RNA secondary structures
Photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) coupled to next-generation sequencing was used to resolve the interaction between the negative-sense RNA genome of IAV and NP during infection of human 293T cells[21]
Summary
Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. We demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells. Influenza A virus (IAV) possesses a segmented, negative-sense RNA genome that is bound by the viral nucleoprotein (NP) throughout replication. Interaction between vRNAs has been demonstrated in vitro and disruption of packaging signals or interacting segment regions attenuated virus replication at the stage of genome packaging[12,13,14]. Viral attenuation is associated with an increase in defective virus production, suggesting that low-NPbinding regions and the predicted RNA structures are required for viral genome packaging
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