{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T04:27:21Z","timestamp":1791433641399,"version":"4.3.3"},"reference-count":60,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T00:00:00Z","timestamp":1771977600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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 \u201cMap-and-Mutate\u201d 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 \u201cMap-and-Mutate\u201d strategy negatively impact viral fitness, underscoring the approach\u2019s 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.<\/jats:p>","DOI":"10.3390\/v18030279","type":"journal-article","created":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T10:59:16Z","timestamp":1772017156000},"page":"279","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["The RNA-Binding Domain NS1 of Influenza as an Antiviral Target: From Evolutionary Conservation Mapping to Experimental Validation"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5915-1186","authenticated-orcid":false,"given":"Luis Andr\u00e9","family":"Santos","sequence":"first","affiliation":[{"name":"Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Sa\u00fade Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016 Lisbon, Portugal"},{"name":"Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003 Lisbon, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jo\u00e3o","family":"Trigueiro-Louro","sequence":"additional","affiliation":[{"name":"Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Sa\u00fade Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016 Lisbon, Portugal"},{"name":"Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003 Lisbon, Portugal"},{"name":"Hospital CUF Tejo Hospital, Av. 24 de Julho 171A, 1350-352 Lisbon, Portugal"},{"name":"Servi\u00e7o de Medicina, Hospital de S\u00e3o Francisco Xavier, Centro Hospitalar Lisboa Ocidental (CHLO), Estrada do Forte do Alto do Duque, 1449-005 Lisbon, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0138-0944","authenticated-orcid":false,"given":"Helena","family":"Rebelo-de-Andrade","sequence":"additional","affiliation":[{"name":"Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Sa\u00fade Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016 Lisbon, Portugal"},{"name":"Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. 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