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Journal of Virology, December 2000, p. 10892-10902, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Phosphorylation Status of the Parvovirus Minute Virus of Mice Particle: Mapping and Biological Relevance of the Major Phosphorylation Sites

Beatriz Maroto, Juan C. Ramírez,dagger and José M. Almendral*

Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas---Universidad Autónoma de Madrid), 28049 Cantoblanco, Madrid, Spain

Received 22 May 2000/Accepted 10 August 2000

The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the 32P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily conserved Ser-2 determining most of the phenotypic effect. In addition, the phosphorylated amino acids were not required for infection initiation or for nuclear translocation of the expressed structural proteins, and thus a role at a late stage of MVMp life cycle is proposed. This study illustrates the complexity of posttranslational modification of icosahedral viral capsids and underscores phosphorylation as a versatile mechanism to modulate the biological functions of their protein subunits.

* 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, December 2000, p. 10892-10902, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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