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

doi:10.1128/JVI.74.8.3804-3814.2000

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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^, Juan C. Ramírez,¹^, 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 analysisin an infectious plasmid. Immunofluorescence of transfected cellsrevealed that the two proteins were involved in cooperative cytoplasmicinteractions for nuclear cotransport. However, while VP-1 translocatedregardless of extension of deletions and did not form capsid epitopesby itself, VP-2 seemed to require cytoplasmic folding and theoverall conformation for nuclear transport. The sequence ₅₂₈KGKLTMRAKLR₅₃₈was found necessary for nuclear uptake of VP-2, even though itwas not sufficient to confer a nuclear localization capacity ona heterologous protein. In the icosahaedral MVMi capsid, thissequence forms the carboxy end of the amphipathic beta-strandI ( $beta$ I), and all its basic residues are contiguously positionedat the face that in the unassembled subunit would be exposed tosolvent. Mutations in singly expressed VP-2 that either decreasethe net basic charge of the exposed face (K530N-R534T), perturbthe 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 residuesclustered at one face of $beta$ I drive VP oligomers into the nucleuspreceding and uncoupled to assembly and that the nuclear environmentis required for MVMi capsid formation in the infected cell. Thedegree of VP-1/VP-2 transport cooperativity suggests that VP trimersare the morphogenetic intermediates translocating through thenuclear pore. The results support a model in which nuclear transportsignaling preserves the VP-1/VP-2 stoichiometry necessary forefficient intranuclear assembly and in which the beta-strandedVP-2 nuclear localization motif contributes to the quality controlof viralmorphogenesis.

^* 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.

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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