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Enteroviruses are one of the most abundant groups of viruses infecting humans, and yet there are no approved antivirals against them. To find effective antiviral compounds against enterovirus B group viruses, an in-house chemical library was screened. The most effective compounds against Coxsackieviruses B3 (CVB3) and A9 (CVA9) were CL212 and CL213, two N -phenyl benzamides. Both compounds were more effective against CVA9 and CL213 gave a better EC 50 value of 1 µM with high a specificity index of 140. Both drugs were most effective when incubated directly with viruses suggesting that they mainly bound to the virions. A real-time uncoating assay showed that the compounds stabilized the virions and radioactive sucrose gradient as well as TEM confirmed that the viruses stayed intact. A docking assay, taking into account larger areas around the 2-and 3-fold axes of CVA9 and CVB3, suggested that the hydrophobic pocket gives the strongest binding to CVA9 but revealed another binding site around the 3-fold axis which could contribute to the binding of the compounds. Together, our data support a direct antiviral mechanism against the virus capsid and suggest that the compounds bind to the hydrophobic pocket and 3-fold axis area resulting in the stabilization of the virion.

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C's cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.