Supplement 2

Enterovirus D68 (EV-D68) causes a diverse range of infectious diseases, especially in children. Until 2010, EV-D68 was primarily associated with mild respiratory diseases. However, since 2010, it has been identified as a cause of severe respiratory illness, requiring hospitalization and intensive care unit admission. The increasing number of acute flaccid paralysis cases linked to EV-D68 infection has become a global public health concern since 2014. No effective vaccines or drugs are currently available for treatment. Through drug screening, we discovered that Geranyl-<i>p-trans-</i>coumaric acid(GCA), a pure compound extracted from Taiwanese propolis, could be a potential inhibitor against EV-D68. The underlying molecular mechanism was determined through a series of <i>in vitro</i> assays. We further confirmed this through drug-resistant virus generation and drug-protein interaction using molecular docking. As a result, GCA directly bound to viral capsid in VP1 at the canyon and heparan sulfate proteoglycan binding sites. This binding did not interfere with viral attachment to the cell surface but specifically inhibited viral uncoating. A single mutation at VP1 T92N was found to affect the binding site, however, it did not confer resistance to GCA. Additionally, we propose that unidentified cellular host factors may be involved in EV-D68 entry, potentially associated with the nonstructural viral protein 2C. Notably, only double mutations (VP1 T92N and 2C K6R) conferred resistance to GCA, suggesting a partial but cooperative role in the antiviral mechanism. The antiviral efficacy of GCA was further confirmed in an animal model. Collectively, our study not only identifies a promising inhibitor but also provides novel mechanistic insights into the viral entry process in EV-D68 life cycle.