AbstractStructure-based mutational analysis of viruses is providing many insights into the relationship between structure and biological function of macromolecular complexes. We have systematically investigated the individual biological roles of charged residues located throughout the structured capsid inner wall (outside disordered peptide segments) of a model spherical virus, the minute virus of mice (MVM). The functional effects of point mutations that altered the electrical charge at 16 different positions at the capsid inner wall were analyzed. The results revealed that MVM capsid self-assembly is rather tolerant to point mutations that alter the number and distribution of charged residues at the capsid inner wall. However, mutations that either increased or decreased the number of positive charges around capsid-bound DNA segments reduced the thermal resistance of the virion. Moreover, mutations that either removed or changed the positions of negatively charged carboxylates in rings of acidic residues around capsid pores were deleterious by precluding a capsid conformational transition associated to through-pore translocation events. The results suggest that number, distribution and specific position of electrically charged residues across the inner wall of a spherical virus may have been selected through evolution as a compromise between several different biological requirements.