We have studied coxsackievirus A9 (CAV9) mutants that each have a single amino acid substitution in the conserved 29-PALTAVETGHT-39 motif of VP1 and a reduced capacity to produce infectious progeny virus. After uncoating, all steps in the infection cycle occurred according to the same kinetics as and similar efficiency to the wild-type virus. However, the particle/infectious unit ratio in the progeny was significantly increased. The differences were apparently due to altered stability of the capsid: there were mutant viruses with enhanced or hindered uncoating, and both of these characteristics were found to reduce fitness under standard passaging conditions. At 32°C the instable mutants had an advantage, while the wild-type and the most stable mutant grew poorly. When comparing the newly published CAV9 structure and the other enterovirus structures, we found that the PALTAVETGHT motif is always in exactly the same position, in a cavity formed by the 3 other capsid proteins, with the C terminus of VP4 between this motif and the RNA. In the 7 enterovirus structures determined to date, the most conserved residues of the studied motif have identical contacts to neighboring residues of VP2, VP3, and VP4. We conclude that (i) the mutations affect the uncoating step necessary for infection, resulting in an untimely or hindered externalization of the VP1 N terminus together with the VP4, and (ii) the reason for the studied motif being evolutionarily conserved is its role in maintaining an optimal balance between the protective stability and the functional flexibility of the capsid.Coxsackievirus A9 (CAV9) is a member of the Enterovirus genus in the family Picornaviridae. Enteroviruses have icosahedral capsids that consist of 60 copies of each of the four capsid proteins VP1 to VP4. Their genomes are single molecules of positive-stranded RNA, approximately 7,500 nucleotides in length.In order to initiate a new infection cycle, enteroviruses have to attach to the host cell, uncoat to reveal the RNA, and at least the RNA must cross a membrane to enter the cell cytoplasm. For CAV9 there are at least two alternative receptors, the integrin ␣ v  3 (25,27), and an unidentified cell surface receptor (17,28). Several other specific receptors are known for various enteroviruses, but the mechanism and location of crossing the membrane are still unclear. It is known that the A particles, altered virus particles lacking the VP4 and having the N terminus of VP1 externalized, of poliovirus type 1 (PV1) are capable of binding to liposomes through the N terminus of VP1 (12). Furthermore, A particles as well as native polioviruses induce channel formation in an artificial membrane (30). The N terminus of VP1 might thus form a pore structure in one of the cell membranes, possibly together with VP4, allowing the RNA to be externalized from the capsid and enter the cytoplasm (12). The pseudoatomic models of the poliovirus 135S and 80S particles were recently determined by cryo-electron microscopy and image reconstruction (5). Based on the...