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, some of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) binds PA and 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 interfere with the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin β RanBP5 and subsequent steps in FluPol assembly. Using FluPol mutants selected after passaging in VHH16-expressing cells, we identified the VHH16 binding site at the interface formed by PA residues with the N-terminus of PB1, overlapping or close to binding sites of two host proteins, ANP32A and RNA-polymerase II RPB1 subunit which are critical for virus replication and transcription, respectively. These data suggest that the VHH16 neutralization is likely due to several activities, altering the import of the PA-PB1 dimer into the nucleus as well as inhibiting specifically virus transcription and replication. Thus, the VHH16 binding site represents a new Achilles' heel for FluPol and as such, a potential target for antiviral development. Author summary: The 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 phages library 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 activity. This nanobody recognizes the PA subunit and the PA-PB1 dimer with high affinity. The VHH binds an epitope overlapping or close to the binding sites of ANP32A and RNA-polymerase II, two key cell proteins involved in FluPol replication and transcription activities, respectively. Furthermore, the VHH was found to interfere with the transport of the PA-PB1 dimer into the nucleus. Thus, the high inhibitory activity of the VHH may result from diverse properties, altering FluPol subunits trafficking inside the cell and FluPol transcription and replication. Targeting the VHH binding site may constitute a powerful strategy to develop new antivirals. [ABSTRACT FROM AUTHOR]