Both a DNA-binding domain and a Vp1 interactive determinant have been mapped to the carboxy-terminal 40 residues of the simian virus 40 (SV40) minor capsid proteins, Vp2 and Vp3 (Vp2/3), with the last 13 residues being necessary for these activities. The role of this DNA-binding domain in SV40 morphogenesis and the ability to separate these two signals were investigated by mutagenesis and assessment of the activity and viability of the mutants. The carboxy-terminal 40 residues of Vp2/3 were expressed as a polyhistidine fusion protein, and five basic residues at the extreme carboxy terminus (Vp3 residues K226, R227, R228, R230, and R233) were mutagenized. The wild-type fusion protein bound DNA with a Kd of 3 x 10(-8) identical to that of the full-length Vp3. Mutant proteins containing either one to three or four amino acid substitutions bound DNA 4- to 7-fold or 20- to 30-fold less well, respectively, than the wild-type protein did. The most severe point mutants showed residual DNA binding similar to that of a truncated protein which lacks the entire 13 carboxy-terminal residues. All of the point mutants were able to interact with Vp1, indicating that the two signals within this region are mediated by different residues. When the mutations were placed into the context of the viral DNA and introduced into cells, all the structural proteins were expressed and localized correctly. Not all, however, were viable: mutant genomes whose Vp2/3 bound DNA with intermediate affinities formed plaques just as well as wild-type SV40 DNA did, but three mutants showing greatly reduced DNA binding failed to form plaques at all. These results are consistent with the hypothesis that Vp2/3 plays an essential role in SV40 virion assembly in the nucleus.