Phosphorylation status of the parvovirus minute virus of mice particle: mapping and biological relevance of the major phosphorylation sites

Abstract

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 (32)P 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.

FIG. 1

Phosphorylation of the MVMp structural proteins assembled in particles. (A) SDS-PAGE of MVMp empty capsids (lanes C) and DNA-full virions (lanes V) purified from infected NB324K cells labeled with [³⁵S]Met-[³⁵S]Cys or [³²P]orthophosphate. Gels were fixed and exposed to autoradiography for 48 h (³⁵S) or blotted to nitrocellulose and the filter was exposed 48 h at −70°C with an intensifying screen (³²P). The positions of the MVMp structural proteins are indicated to the left, and their approximate molecular weights (MW) are given to the right in kilodaltons (B) Phosphoamino acid composition of the VP-1 and VP-2 proteins of MVMp capsid. Shown are autoradiograms of thin-layer 2D electrophoresis of phosphoamino acid analysis of labeled VP-1 and VP-2. The regions circled by dashed lines indicate the positions where the phosphoamino acid markers migrated. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine. 1D, first dimension; 2D, second dimension.

FIG. 2

2D phosphopeptide maps of parvovirus MVMp capsid proteins. VP-1 and VP-2 proteins isolated from purified ³²P-labeled MVMp capsids were digested with proteases and subjected to two-dimensional TLC analysis. Shown are the phosphopeptide maps from trypsin (T), trypsin-plus-endoproteinase-V8 (T+V8), and trypsin-plus-chymotrypsin (T+QT) digestions. The primary VP-2 tryptic peptides are alphabetically designated in the autoradiograms according to their 2D migration (left to right starting from the bottom of the plates), and this nomenclature is maintained for the peptides shared with VP-1 (D to O). Specific VP-1 peptides were designated P to S. Peptides arising from subsequent digestions were identified by comparative analysis of superimposed films and are designated by the initial letter of the secondary protease used (V, V8; Q, chymotrypsin) followed by an arbitrary number. Plates were exposed to autoradiography with an intensifying screen for 4 to 7 days at −70°C. Only the areas of the plates where the phosphopeptides migrated are shown. 1D, first dimension; 2D, second dimension; o, origin. The resolution is indicated by the scale bar.

FIG. 3

Analysis of phosphopeptide B localization in VP-2. (A) 2D tryptic maps of [³⁵S]Met-[³⁵S]Cys-labeled VP-2 and VP-3 proteins isolated from purified MVMp virions. The dried TLC plates were exposed for autoradiography in a Fujix Bas 1000 phosphorimager (Fuji) for 5 days. The arrow indicates the absence in VP-3 fingerprint of VP-2 peptide B. 1D, first dimension; 2D, second dimension; o, origin. (B) In vitro trypsin digestion of MVMp particles. Purified ³⁵S- and ³²P-labeled MVMp empty capsids and DNA-containing viruses were digested (+) with an excess of trypsin for 2 h at 37°C or not digested (−), and the samples were resolved by SDS-PAGE (10% polyacrylamide) and blotted to nitrocellulose membranes, and the filters were exposed for autoradiography in a phosphorimager for 2 days. The positions of the three MVMp structural proteins are indicated.

FIG. 4

The VP-2 N-terminal domain is phosphorylated exclusively in serine residues. (A) Phosphoamino acid analysis of peptide B. Autoradiography of acid hydrolysis of peptide B subjected to one-dimensional thin-layer electrophoresis is shown. The positions where markers migrated are encircled by dotted lines. T+Y, phosphothreonine plus phosphotyrosine; S, phosphoserine. (B) Quantitative analysis of the proportion of the peptide B phospho-label corresponding to phosphoamino acids. ND, undigested peptide B; D, peptide B digested to amino acids with proteinase K. Markers and electrophoretic conditions were as in panel A. Both type of analyses were performed with approximate 200 cpm of pure phosphopeptide B, and the plates were exposed for autoradiography in a phosphorimager (Fuji) for seven days. o, origin.

FIG. 5

Several serine residues are phosphorylated in the VP-2 N-terminal domain. The figure shows VP-2 tryptic maps of ³²P-labeled MVMp capsids purified from the infection of NB324K cells with the indicated virus mutants. Note the absence of phosphopeptide B in the autoradiogram of the multiple mutant 4S/G (arrowhead) and its comparable signal in the single-site mutants shown (S2G and S10G). Plates were exposed in a phosphorimager (Fuji) for 4 days. 1D and 2D, first (electrophoresis) and second (chromatography) dimensions, respectively; o, origin.

FIG. 6

Mapping the phosphorylated serine residues within the VP-2 N-terminal domain. Peptide B isolated from VP-2 phosphopeptide maps of wt and mutant capsids (Fig. 2 and 5) were incubated with the indicated proteases, and the digestion products were resolved by one-dimensional thin-layer electrophoresis. Samples were applied at the center of the TLC plates. Arrowheads in the panels point to mutant phosphopeptides not seen in the corresponding protease digestions of wt peptide B. Abbreviations: ND, undigested peptide B; Q, α-chymotrypsin; TM, thermolysin; V8, endoproteinase Glu-C. The amino acid sequence of wt tryptic peptide B is shown at the bottom left with the phosphoserine residues determined in this work encircled in bold and the protease cleavage sites indicated by arrows. Black arrows: protease recognition sites refractory to cleavage; grey arrows, cleavage sites accessible to proteases. The single-letter code for the amino acids is as follows: A, Ala; D, Asp; E, Glu; G, Gly; H, His; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val.

FIG. 7

VP-2 to VP-3 conversion in MVMp virus lacking VP-2 N-terminal phosphorylation. (A) Scheme of VP-2 and VP-3 proteins of MVMp with the trypsin-sensitive sequence (64, 71) and the three mapped VP-2 phosphoserine residues highlighted. (B) SDS-PAGE of ³⁵S-labeled structural proteins of wt and 4S/G mutant virions purified from NB324K cultures at 72 h p.i., showing extensive processing of the VP-2 subunits. (C) Quantitative analysis of VP-2 exposure in immature viruses. ³⁵S-labeled virions (0.01 μg) purified at 16 h p.i. were digested with the indicated amounts of trypsin (in micrograms) for 30 min at 37°C and analyzed by SDS-PAGE. The position of the VP-3 protein is indicated. A total of 500 cpm was loaded per sample, and exposure was for 4 days in a Fujix Bas 1000 phosphorimager (Fuji). The amino acid code is as in Fig. 6.

FIG. 8

Phosphorylation of the VP-2 N terminus is required late in the MVMp infection cycle. (A) Plaque-forming capacity of wt and VP-2 phosphorylation mutants. Monolayers of NB324K cells were inoculated with serial dilutions of the indicated viruses and the number of plaques was scored from duplicate plates. Bars represent the mean and standard error of the mean from three independent experiments. (B) Plaque morphology of MVMp viruses in NB324K cells. Plates were developed 6 days after inoculation. Examples of viruses with normal (wt and S6G) and small (S2G and 4S/G) plaque sizes are shown. (C) Assessment of the capacity of the mutant viruses to initiate infection. NB324K cells were inoculated with normalized amounts of purified virus particles, and the number of positive cells for capsid protein synthesis was determined by IF at 24 h p.i. The mean numbers of scored cells and standard errors of the mean from three experiments are shown. (D) Subcellular localization of the synthesized capsid proteins. NB324K cells were inoculated with the indicated viruses and stained by IF at 24 h p.i. with an MVMp capsid antiserum.