Antiviral activity of intracellular nanobodies targeting the influenza virus RNA-polymerase core

Abstract

Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, several of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) targets PA, but preferentially bind the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to impair the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin b RanBP5 and subsequent steps in FluPol assembly. These data suggest that the VHH16 neutralization activity is likely due to an alteration of the import of the PA-PB1 dimer into the nucleus, resulting to an inhibition of FluPol functioning. VHH16 binding site represent a potential target for antiviral development.