Structural characterization of Posaconazole- parechovirus A3 capsid interaction using molecular simulations and cell infection assays

Parechovirus-A3 (PeV-A3) is a major cause of central nervous system infections in neonates, associated with meningoencephalitis and neurodevelopmental delays. Yet there are no specific antivirals against PeV-A3 infection. A newer clade of PeV-A3 has emerged post COVID-19, and our preliminary data shows that infants are not immune to the emerging variants, underscoring the need for a focused therapeutic discovery. Posaconazole (POS) is seen to attenuate PeV-A3 entry and infection, but the mechanistic understanding of drug-capsid interaction remains unknown. To this end, we combined mechanistic interrogation of drug-capsid interactions and conformational dynamics, with experimental infection assays. We identify two POS binding sites: a VP0-VP1 interface in the 2009 isolate (US WI-09), and a VP3-VP1 groove in the 2022 isolate (9C-CMKC) and a laboratory-selected POS-resistant variant (US WI-09 POSA P14). Apo simulations showed congruent trajectories of POS: to the VP0 face in US WI-09, and to the VP3 arm in 9C-CMKC and US WI-09 POSA P14. The relative contribution of VP0 to the drug-capsid interaction decreased from 86% in US WI-09 to zero in both 9C-CMKC and US WI-09 POSA P14, indicating a redistribution of binding interactions across capsid subunits. Cysteine or arginine substitutions to VP1 Tyr224 abolished the Tyr224-VP3 anchor. Functionally, 0.3 {micro}M POS inhibited replication of US WI-09 and 9C-CMKC in Vero-P cells but only US WI-09 in receptor-overexpressing Vero-MYADM cells. These findings suggest that POS efficacy depends on binding-site localization near the MYADM-engaging VP0 interface, revealing how emerging PeV-A3 variants may retain drug binding while evading antiviral inhibition.