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Journal of Virology, August 2001, p. 7321-7329, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7321-7329.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Peggy P. Li, Akira Nakanishi, Dorothy Shum, Peter C.-K. Sun, Adler M. Salazar, Cesar F. Fernandez, Sze-Wai Chan, and Harumi Kasamatsu*

Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095

Received 19 January 2001/Accepted 17 May 2001

A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1Delta C58 pentamer bound DNA with a Kd of 1.8 × 10-9 M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1Delta C17 had comparable apparent Kds of 5.3 × 10-9 to 7.3 × 10-9 M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1Delta C58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1 · 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1Delta C58 level for NLSm1 · 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209-8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-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, NLSm2, and NLSm1 · 2 replicated normally following transfection into host cells 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 nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.

* Corresponding author. Mailing address: Molecular Biology Institute, 456 Boyer Hall, University of California at Los Angeles, 611 E. Charles E. Young Dr., Box 951570, Los Angeles, CA 90095-1570. Phone: (310) 825-3048. Fax: (310) 206-7286. E-mail: harumi_K{at}mbi.ucla.edu.

Journal of Virology, August 2001, p. 7321-7329, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7321-7329.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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