Inhibition of influenza virus replication by artificial proteins (αReps) targeting its RNA-polymerase

Seasonal epidemics and pandemics caused by influenza A viruses still represent a main public health burden in the world. Influenza viruses replicate and transcribe their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol) through extensive structural rearrangements and differential interactions with host cell factors. To get insights into its functioning, we screened a phage-display library of biosynthetic proteins (named Reps and build on a rigid alpha-helicoidal HEAT-like scaffold) against the structurally invariant FluPol core and several flexibly-linked domains of the FluPol PB2 subunit. Several Reps specific of the cap binding domain [CBD], the 627-domain and the NLS domain of the PB2 FluPol subunit displayed FluPol inhibitory and virus neutralization activities when transiently expressed in the cytosol. Furthermore, intracellular ectopic inducible expression of the Reps C3 and F3 (specific of the CBD and the 627-domain, respectively) in influenza virus permissive cells blocked transcription and multiplication of viruses representative of the H1N1, H3N2 and H7N1 subtypes, even when induced at late times post-infection. A synergic inhibitory effect on FluPol activity and virus multiplication was evidenced when the two Reps were covalently linked. These results suggest that i) interfering with FluPol structural rearrangements that are concomitant to its various activities may represent a promising strategy to block virus multiplication and to design new types of antivirals such as dual binders targeting distant sites on FluPol and ii) the 627-domain could be efficiently targeted to design influenza antivirals. Author SummaryThe influenza virus RNA-polymerase (FluPol) ensures genome transcription and replication in the nucleus of the infected cells. To select ligands able to interfere with FluPol functions, we screened a library of phages encoding biosynthetic proteins (named Reps) for binding to FluPol subunits and domains. When expressed intracellularly, several of them display efficient FluPol blocking and virus neutralizing activities. Reps C3 and F3 assembled through covalent linkages blocked FluPol activity more efficiently than their precursors. These Reps impaired multiplication of H1N1, H3N2 and H7N1 viruses, showing that their binding sites may constitute effective targets for new antiviral development.