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Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReED\u2019s mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity\/resistance. Our data overall suggest that the ReED in NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and\/or polymerase, thereby likely facilitating initiation of RNA synthesis.<\/jats:p>","DOI":"10.1371\/journal.ppat.1014239","type":"journal-article","created":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T17:41:37Z","timestamp":1785951697000},"page":"e1014239","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["A novel dimerization site in non-structural protein 5A of hepatitis C virus regulates viral replication 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