Genotype-by-Inhibitor Interactions to Dissect Enterovirus Replication

Genotype-by-Inhibitor Interactions to Dissect Enterovirus Replication | 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 Genotype-by-Inhibitor Interactions to Dissect Enterovirus Replication Patrick Dolan, William Bakhache, Walker Symonds-Orr This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7660613/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Replication organelles of positive-sense RNA viruses are essential to virus biology, yet their molecular mechanisms remain poorly defined. While deep mutational scanning (DMS) measures the impact of mutations across viral proteins, it cannot resolve their effects on specific functions. Here, we present a strategy to integrate mutational scanning in the context of specific virus- and host-targeted inhibitors with structural modeling to dissect structural and mechanistic details of Enterovirus replication. Our results reveal key insights into the function of nonstructural proteins in the context of viral replication. We use this approach to clarify the modular architecture of the viral 2C protein, dissecting the functional partitioning of its ‘virus-facing’ cytoplasmic, enzymatic domain from ‘host-facing’ functions at membrane-binding domain, revealing evidence for computationally-predicted structural transitions associated with host protein binding. We further show that inhibition of the 3C protease enriches for mutations in 2A, highlighting compensatory crosstalk between viral proteases. Finally, targeting host phospholipid synthesis triggers a dose-dependent shift in mutational tolerance in the viral 3A protein, showing how preference for distinct interaction interfaces with either a host enzyme or its adapter protein varies across inhibitory environments. Our approach, which quantifies the impact of pharmacological probes on viral fitness by comprehensive mutational scanning, creates a virtuous cycle where DMS validates and refines structural predictions that, in turn, serve to contextualize mutational data, all toward a more complete model of positive-sense RNA virus replication. Biological sciences/Microbiology/Virology/Virus–host interactions Biological sciences/Genetics/Evolutionary biology Biological sciences/Computational biology and bioinformatics/Protein structure predictions Positive-sense RNA viruses Enteroviruses Replication organelles Deep mutational scanning Pharmacological perturbations Structural modeling Virus–host interactions Full Text Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review 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. 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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-7660613","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":521293549,"identity":"1158681f-9bdb-448f-b862-8864f792d8a7","order_by":0,"name":"Patrick 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