Simian virus 40 Vp1 DNA-binding domain is functionally separable from the overlapping nuclear localization signal and is required for effective virion formation and full viability

A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major c apsid protein Vp1, and the domain's function in the SV40 life cycle was exa mined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1 Delta C 58 pentamer bound DNA,with a K-d of 1.8 X 10(-9) M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1 Delta C17 had comparable apparent K(d)s of 5.3 X 10(-9) to 7.3 X 10(-9) M in ter ms of the protein monomers. Previously identified on Vp1 was a nuclear loca lization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4- KRK-6) and NLS2 (15-KKPK-18). Vp1 Delta C58 pentamers harboring multiple-po int mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1 . 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1 Delta C58 level for NLSm1 . 2 Vp1. These data, along with those of N-te rminally truncated proteins, placed the DBD in overlap with the bipartite N LS. The role of the Vp1 DBD during infection was investigated by taking adv antage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada , M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209-8216, 1994), in whic h an NLS-defective Vp1 could localize to the nucleus in the presence of wil d-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlaping SV40 (NO-SV40) DNA NLSm1, NLS m2, and NLSm1 - 2 replicated normally following transfection into host cell s and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1 - 2 were nonviab le, and the mutant Vp1s unexpectedly failed to localize to the nucleus thou gh Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant si gnal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the m utant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, di splayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected c ell lysate revealed a 10-fold reduction in the level of DNase I-protected v iral DNA, and yet virion-like particles were found among the DNase I-resist ant material. Collective results support a role for Vp1 NLS2-DBD2 in the as sembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.