Comparative Analysis of Deep Mutational Scanning Datasets in Enteroviruses A and B Identifies Functional Divergence and Therapeutic Targets

Comparative Analysis of Deep Mutational Scanning Datasets in Enteroviruses A and B Identifies Functional Divergence and Therapeutic Targets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparative Analysis of Deep Mutational Scanning Datasets in Enteroviruses A and B Identifies Functional Divergence and Therapeutic Targets Patrick Dolan, Beatriz Álvarez-Rodríguez, William Bakhache, Lauren McCormick, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7483105/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2026 Read the published version in Nature Ecology & Evolution → Version 1 posted You are reading this latest preprint version Abstract Deep mutational scanning (DMS) can define functional constraints acting on viral proteomes by quantifying the effects of mutations on viral fitness. However, DMS analyses do not discern type-specific from species-level constraints, limiting their utility in understanding how selective pressures change as viral families diversify. Here, we show that comparison of DMS datasets from related viruses can overcome these limitations. By contrasting two proteome-wide DMS datasets from prototypical members of the enterovirus A and B species, we identify evolutionary constraints at the species level to occur across core enzymatic machinery and capsid assembly interfaces. In contrast, type-level constraints are observed across host-interaction sites in both structural and non-structural proteins. Furthermore, we find DMS data to reflect both type- and species-level evolutionary signatures in nature yet diverge at conserved hotspots subjected to selection pressures that are lacking in vitro. Finally, we highlight the utility of comparative DMS studies for drug discovery by identifying a novel, mutationally constrained pocket in the 2C helicase that is conserved across all major human enterovirus species. Collectively, our findings provide a framework for dissecting evolutionary pressures acting at different evolutionary scales and for guiding the rational design of broad-spectrum therapeutics with high barriers to resistance. Biological sciences/Microbiology/Virology/Viral evolution Biological sciences/Computational biology and bioinformatics/High-throughput screening enteroviruses enterovirus A71 coxsackievirus B3 deep mutational scanning drug discovery Full Text Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Published Journal Publication published 23 Feb, 2026 Read the published version in Nature Ecology & Evolution → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7483105","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":510473158,"identity":"d1235d4b-6d81-4a7c-8e8e-995a0a35b761","order_by":0,"name":"Patrick Dolan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYLCCBCBkOMDcwMBQwcBjQFg9M0wLI1DLGWK1MMC0MLYxMBDUYs5+/tiHBzVpDHzHGxs/F867I2POwH7xMQ8eLZY9ycwzEo7lMEieOdgsPXPbMx7LBp5iY3xaDA4kMzMkNlQwGNxIbJDm3XaYx+AAT5rkDHxazj+Ga2n+zTuHGC03wLbkgLS0SfM2gLSwH5P4gM8vMx4bMyQcS+MB+qXNmucYUMthHmYDfFrM+RMfM/6oSZbjO958+DZPzWF7g+PtDx8k4HMYlEYKImYCsYlNlv0BXi2jYBSMglEw4gAASeZOjNDqskkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4169-0058","institution":"NIH-NIAID Division of Intramural Research","correspondingAuthor":true,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Dolan","suffix":""},{"id":510473159,"identity":"971b0cef-5922-444c-bf64-08c68ef72c56","order_by":1,"name":"Beatriz Álvarez-Rodríguez","email":"","orcid":"https://orcid.org/0000-0002-9898-4997","institution":"Institute for Integrative Systems Biology (I2SysBio) - 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