The RNA-Binding Domain NS1 of Influenza as an Antiviral Target: From Evolutionary Conservation Mapping to Experimental Validation

Non-structural protein 1 (NS1) of influenza A virus is a multifunctional virulence factor and represents a promising anti-influenza target, considering its conserved and druggable structure. As antiviral target, NS1-RNA-binding domain (RBD) remains unexplored, despite its critical role in replication. In this study, we applied a "Map-and-Mutate" strategy to identify and functionally validate highly conserved and druggable regions within the NS1-RBD. Using large-scale sequence alignments and structural characterization, we integrated conservation and druggability analyses to predict conserved druggable pockets and top-ranked hot spots, mutate the five most promising residues (L15, W16, R19, R35, and L43) and study their impact on viral fitness. In vitro, the mutations W16 and R35 caused most significant reduction in viral fitness; however, L15 and R19 also impaired replication. Combined mutations involving W16 and either L15 or L43 exerted a cumulative effect, reducing viral replication, hemagglutination titers and neuraminidase activity. This study demonstrates that most residues identified and investigated using the "Map-and-Mutate" strategy negatively impact viral fitness, underscoring the approach's value in pinpointing novel antiviral targets. Together with our prior research, this study reinforces the importance of NS1 as a promising antiviral target, providing a rationale for designing and developing therapies with a higher resilience to viral resistance.