We describe a structural rearrangement that can occur in parvovirus minute virus of mice (MVMp) virions following prolonged exposure to buffers containing 0.5 mM EDTA. Such particles remain stable at 4 degrees C but undergo a conformational shift upon heating to 37 degrees C at pH 7.2 that leads to the ejection of much of the viral genome in a 3'-to-5' direction, leaving the DNA tightly associated with the otherwise intact capsid. This rearrangement can be prevented by the addition of 1 mM CaCl(2) or MgCl(2) prior to incubation at 37 degrees C, suggesting that readily accessible divalent cation binding sites in the particle are critical for genome retention. Uncoating was not seen following the incubation of virions at pH 5.5 and 37 degrees C or at pH 7.2 and 37 degrees C in particles with subgenomic DNA, suggesting that pressure exerted by the full-length genome may influence this process. Uncoated genomes support complementary-strand synthesis by T7 DNA polymerase, but synthesis aborts upstream of the right-hand end, which remains capsid associated. We conclude that viral genomes are positioned so that their 3' termini and coding sequences can be released from intact particles at physiological temperatures by a limited conformational rearrangement. In the presence of divalent cations, incremental heating between 45 degrees C and 65 degrees C induces structural transitions that first lead to the extrusion of VP1 N termini, followed by genome exposure. However, in cation-depleted virions, the sequence of these shifts is blurred. Moreover, cation-depleted particles that have been induced to eject their genomes at 37 degrees C continue to sequester their VP1 N termini within the intact capsid, suggesting that these two extrusion events represent separable processes.