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

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 {beta} 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. Author SummaryThe influenza virus RNA-polymerase (FluPol) ensures genome transcription and replication in the nucleus of the infected cells. To select ligands able to block FluPol activities, we screened a library of phages encoding nanobodies and resulting from the immunization of a llama with FluPol subunits. When expressed intracellularly, one of the nanobodies displays highly efficient FluPol blocking and virus neutralizing activities. This nanobody binds FluPol with high affinity and recognizes preferentially the PA-PB1 assembled subunits. Furthermore, it was found to interfere with the transport of the PA-PB1 dimer into the nucleus, suggesting that targeting FluPol trafficking between the cytoplasm and the nucleus may constitute a powerful strategy to develop new antivirals.