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 αReps and built 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 αReps specific of the cap binding domain [CBD], the 627-domain and the NLS domain of PB2 displayed FluPol inhibitory and virus neutralization activities when transiently expressed in the cytosol. Furthermore, intracellular ectopic inducible expression of the αReps C3 and F3 (specific of the CBD and the 627-domain, respectively) in influenza virus permissive cells blocked multiplication of viruses representative of the H1N1, H3N2 and H7N1 subtypes, even when induced at late times post-infection. Bispecific αReps constructs (C3-F3 and F3-C3) display a higher FluPol inhibitory activity than their monomeric counterparts. These results suggest that interfering with FluPol structural rearrangements 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. Furthermore, we found that the 627-domain constitutes a new possible target for engineering influenza antivirals.