Abstract: Cellular and Molecular Life Sciences (2022) 79:227 https://doi.org/10.1007/s00018-022-04246-w Cellular and Molecular Life Sciences ORIGINAL ARTICLE Impairment of SARS‑CoV‑2 spike glycoprotein maturation and fusion activity by nitazoxanide: an effect independent of spike variants emergence Anna Riccio1 · Silvia Santopolo1 · Antonio Rossi2 · Sara Piacentini1 · Jean‑Francois Rossignol3 · M. Gabriella Santoro1,2 Received: 5 January 2022 / Revised: 3 March 2022 / Accepted: 11 March 2022 / Published online: 7 April 2022 © The Author(s) 2022, corrected publication 2022 Abstract SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here, we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell–cell fusion assays, we show that nitazoxanideinduced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell–cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID19 infections. Keywords Broad-spectrum antiviral · COVID-19 · Nitazoxanide · SARS-CoV-2 variants · SARS-CoV-2 spike · Syncytia formation