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Journal of Virology, April 2000, p. 3804-3814, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Beta-Stranded Motif Drives Capsid Protein Oligomers of the Parvovirus Minute Virus of Mice into the Nucleus for Viral Assembly

Eleuterio Lombardo,1,dagger Juan C. Ramírez,1,dagger Mavis Agbandje-McKenna,2 and José M. Almendral1,*

Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas---Universidad Autónoma de Madrid), 28049 Cantoblanco, Madrid, Spain,1 and Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom2

Received 18 October 1999/Accepted 3 January 2000

The determinants of nuclear import in the VP-1 and VP-2 capsid proteins of the parvovirus minute virus of mice strain i (MVMi) synthesized in human fibroblasts were sought by genetic analysis in an infectious plasmid. Immunofluorescence of transfected cells revealed that the two proteins were involved in cooperative cytoplasmic interactions for nuclear cotransport. However, while VP-1 translocated regardless of extension of deletions and did not form capsid epitopes by itself, VP-2 seemed to require cytoplasmic folding and the overall conformation for nuclear transport. The sequence 528KGKLTMRAKLR538 was found necessary for nuclear uptake of VP-2, even though it was not sufficient to confer a nuclear localization capacity on a heterologous protein. In the icosahaedral MVMi capsid, this sequence forms the carboxy end of the amphipathic beta-strand I (beta I), and all its basic residues are contiguously positioned at the face that in the unassembled subunit would be exposed to solvent. Mutations in singly expressed VP-2 that either decrease the net basic charge of the exposed face (K530N-R534T), perturb the hydrophobicity of the opposite face (L531E), or distort the beta I conformation (G529P) produced cytoplasmic subviral oligomers. Particle formation by beta I mutants indicated that the basic residues clustered at one face of beta I drive VP oligomers into the nucleus preceding and uncoupled to assembly and that the nuclear environment is required for MVMi capsid formation in the infected cell. The degree of VP-1/VP-2 transport cooperativity suggests that VP trimers are the morphogenetic intermediates translocating through the nuclear pore. The results support a model in which nuclear transport signaling preserves the VP-1/VP-2 stoichiometry necessary for efficient intranuclear assembly and in which the beta-stranded VP-2 nuclear localization motif contributes to the quality control of viral morphogenesis.

* Corresponding author. Mailing address: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain. Phone: 34-91-3978048. Fax: 34-91-3978087. E-mail: JMAlmendral{at}cbm.uam.es.

dagger Present address: Centro Nacional de Biotecnología (CSIC), 28049 Cantoblanco, Madrid, Spain.

Journal of Virology, April 2000, p. 3804-3814, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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