© 2015 The Authors. Small spherical viruses are paradigms of supramolecular self-assembly. Identifying the specific structural determinants for virus assembly provides guidelines to develop new antiviral drugs or engineer modified viral particles for medical or technological applications. However, very few systematic studies have been carried out so far to identify those chemical groups at interfaces between virus capsid subunits that are important for viral assembly and function. Foot-and-mouth disease virus (FMDV) and other picornaviruses are assembled in a stepwise process in which different protein–protein interfaces are formed: 5 protomeric subunits oligomerize to form a pentameric intermediate, and 12 of these stable pentameric building blocks associate to form a labile capsid. In this study, a systematic mutational analysis revealed that very few amino acid side chains involved in substantial interactions between protomers within each pentamer are individually required for virus infectivity. This result contrasts sharply with the previous finding that most amino acid side chains involved in interactions between pentamers during the next assembly step are individually required for infectivity. The dramatic difference in sensitivity to single mutations between the two types of protein–protein interfaces in FMDV is discussed in terms of possible structural strategies for achieving self-assembly and genome uncoating in the face of diverse selective constraints. ; This work was funded by grants from the Spanish Government (BIO2009-10092 and BIO2012-37649) and Comunidad de Madrid (S-505/MAT-0303) to M. G. M., and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa> Peer Reviewed