In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.Many fundamental biological processes involve protein-nucleic acid recognition. Double-stranded DNA-protein interactions have been extensively studied, but single-stranded (ss) 1 DNA-or RNA-protein interactions are much less characterized. In particular, very little is known about the molecular interactions between the nucleic acid genome of ssDNA or RNA virus and its protein shell, or the functional role of those interactions.Such knowledge may be essential for a better understanding of the life cycle of viruses, and for the design of drugs aimed at disrupting nucleic acid-protein interfaces.The structure of a helical virus, tobacco mosaic virus, has revealed defined interactions between repeating sets of three nucleotides in its ssRNA genome and each capsid subunit (reviewed in Ref. 1). In the crystal structures of spherical viruses (reviewed in Refs. 2-6) a large part, or all, of the nucleic acid component is invisible, because it is randomly oriented within the viral particles that form the crystal. Fortunately, in a few icosahedral viruses some segments of the nucleic acid molecule (ϳ10 -60% of the total) are arranged with the same symmetry as the capsid, and could be visualized. The structural analysis (reviewed in Refs. 4, 7-10) has revealed that the visible capsidnucleic acid interfaces generally include a limited number of amino acids and some 7-30 nucleotides that in several, but not in all cases, form intramolecular double-helical segments. The tertiary structure acqui...