Hydroxyproline in the major capsid protein VP1 of polyomavirus.

Amino acid analysis of [3H]proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of 3H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from [35S]methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.     

Conformational changes of polyomavirus major capsid protein VP1 in sodium dodecyl sulfate solution.

Conformations of polyomavirus (Py) major capsid protein VP1 were analyzed by circular dichroism (CD) and fluorescence spectroscopy in the presence of sodium dodecyl sulfate (SDS). Binding of PyVP1 to SDS induced marked conformational changes of PyVP1, which were reflected on the CD and fluorescence spectra. Abrupt changes in both optical properties occurred within the narrow ranges of SDS concentrations with the transition midpoints closely related to SDS micelle formation. Analysis of circular dichroism spectra showed that the contents of alpha-helices, beta-sheets, beta-turns and random coils in PyVP1 varied upon addition of SDS, demonstrating the exquisite sensitivity of the conformations of the protein to the environment. The interactions of PyVP1 with SDS were shown to be dependent on the ionic strength of the protein solution, suggesting that both hydrophobic and electrostatic forces contribute to the PyVP1-SDS complex formation. The SDS-induced conformational changes of PyVP1 appeared to be a two-stage process.     

Synthesis, posttranslational modifications, and nuclear transport of polyomavirus major capsid protein VP1.

Polyomavirus major capsid protein VP1 synthesis was studied in infected primary baby mouse kidney cells. A standard curve of VP1 protein was used to quantitate VP1 in the cytoplasm and nucleus of infected cells during the time course of infection. Polyomavirus VP1 continued to be accumulated in the cytoplasm of the cells until 27 h postinfection, at which time the synthesis of VP1 leveled off. VP1 continued to accumulate in the nucleus of the infected cells throughout the course of infection. The presence of the six isospecies, A to F, of polyomavirus VP1 was also studied to determine the relative quantity of each species during the time course of infection. All six species were found in the cytoplasm and nucleus of infected cells at various times postinfection. However, the relative quantity of each species was different at early as compared with later times of infection. In addition, phosphorylated VP1 was found in isolated polyribosomes of infected cells, suggesting that phosphorylation of VP1 is a cotranslational modification. Examination of the effect of macromolecular synthesis on the transport of VP1 into the nucleus of infected baby mouse kidney cells as well as the rate of its nuclear accumulation during and after protein synthesis inhibition revealed that the continual transport and accumulation of VP1 in the nucleus required protein synthesis.     

The polyomavirus major capsid protein VP1 interacts with the nuclear matrix regulatory protein YY1

Polyomavirus reaches the nucleus in a still encapsidated form, and the viral genome is readily found in association with the nuclear matrix. This association is thought to be essential for viral replication. In order to identify the protein(s) involved in the virus-nuclear matrix interaction, we focused on the possible roles exerted by the multifunctional cellular nuclear matrix protein Yin Yang 1 (YY1) and by the viral major capsid protein VP1. In the present work we report on the in vivo association between YY1 and VP1. Using the yeast two-hybrid system we demonstrate that the VP1 and YY1 proteins physically interact through the D-E region of VP1 and the activation domain of YY1.     

Polymorphism in the assembly of polyomavirus capsid protein VP1.

Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid.     

Localization of calcium on the polyomavirus VP1 capsid protein.

Our laboratory has previously shown that the divalent cation Ca2+ is an integral part of the polyomavirus and plays a major role in stabilizing the intact virion structure. In this report, we show that calcium is sequestered on the major capsid protein VP1 of polyomavirus. The virion structural proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being transferred to nitrocellulose and probed with 45CaCl2. Autoradiography revealed 45Ca binding exclusively to VP1. Increasing the amount of VP1 transferred to the nitrocellulose resulted in a concomitant increase in 45Ca binding. 45Ca binding to VP1 could be reduced by competition with an excess of unlabeled CaCl2. Separation of the species of VP1 by two-dimensional gel electrophoresis before electroblotting and probing with 45CaCl2 revealed that all six species (A to F) bind the radiolabeled calcium. Formic acid cleavage of the 43-kilodalton (kDa) VP1 protein into 29-, 18-, and 16-kDa fragments before 45Ca-binding analysis revealed that only the 18- and 16-kDa carboxyl-terminal fragments of this protein bind 45Ca.     

Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1.

Polyomavirus normally assembles in the nucleus of infected mouse cells. Sf9 insect cells expressing the polyomavirus major capsid protein VP1 were examined by electron microscopy. Capsidlike particles of apparently uniform size were found in the nucleus. Immunogold electron microscopy demonstrated abundant VP1 in the cytoplasm which was not assembled into any recognizable higher-order structure. Cytoplasmic VP1 assembled after the cells were treated with the calcium ionophore ionomycin. Purified VP1 aggregates were shown by negative staining and cryoelectron microscopy to consist predominantly of particles similar to the empty T = 7 viral capsid. Thus, polyomavirus VP1 can assemble in vivo into capsids independent of other viral proteins or DNA. Nuclear assembly may result from increased available calcium in this subcellular compartment.     

Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry.

A complex of the polyomavirus internal protein VP2/VP3 with the pentameric major capsid protein VP1 has been prepared by co-expression in Escherichia coli. A C-terminal segment of VP2/VP3 is required for tight association, and a crystal structure of this segment, complexed with a VP1 pentamer, has been determined at 2.2 A resolution. The structure shows specific contacts between a single copy of the internal protein and a pentamer of VP1. These interactions were not detected in the previously described structure of the virion, but the location of VP2 in the recombinant complex is consistent with features in the virion electron-density map. The C-terminus of VP2/VP3 inserts in an unusual, hairpin-like manner into the axial cavity of the VP1 pentamer, where it is anchored strongly by hydrophobic interactions. The remainder of the internal protein appears to have significant flexibility. This structure restricts possible models for exposure of the internal proteins during viral entry.     

Comparison of nonphosphorylated and phosphorylated species of polyomavirus major capsid protein VP1 and identification of the major phosphorylation region.

The major virion protein of polyomavirus, VP1, consists of about six isoelectric species designated A through F. The minor species D, E, and F are phosphorylated and are thought to serve as viral receptors. We first wanted to distinguish whether all VP1 species are derived by post-translational modification from a common amino acid sequence or whether one or more of the species contain a region(s) of altered amino acid sequence resulting from alternate mRNA processing. We compared the VP1 species by detailed peptide mapping with several combinations of specific protease and radioisotopic labels. This approach enabled us to examine more than 80% of the predicted VP1 sequence, including the amino-and carboxy-termini. We found no evidence of sequence differences among any of the VP1 species. The specific incorporation of 32Pi was found to be the same for all of the phosphorylated species. Comparison of the phosphorylation sites of in vivo 32Pi-labeled D, E, and F by peptide mapping showed them to be identical. Each phosphorylated species contained a single major phosphopeptide and several minor phosphopeptides. The major phosphoamino acid, identified by acid hydrolysis, was phosphothreonine, with phosphoserine also present. By using chemical cleavage methods, we localized the major phosphorylation region to a central portion of the VP1 sequence. We discuss some features of this region and relate this information to functional implications of phosphorylation.     

Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1.

The major capsid protein of polyomavirus, VP1, has been expression cloned in Escherichia coli, and the recombinant VP1 protein has been purified to near homogeneity (A. D. Leavitt, T. M. Roberts, and R. L. Garcea, J. Biol. Chem. 260:12803-12809, 1985). With this recombinant protein, a nitrocellulose filter transfer assay was developed for detecting DNA binding to VP1 (Southwestern assay). In optimizing conditions for this assay, dithiothreitol was found to inhibit DNA binding significantly. With recombinant VP1 proteins deleted at the carboxy and amino termini, a region of the protein affecting DNA binding was identified within the first 7 amino acids (MAPKRKS) of the VP1 amino terminus. Southwestern analysis of virion proteins separated by two-dimensional gel electrophoresis demonstrated equivalent DNA binding among the different VP1 isoelectric focusing subspecies, suggesting that VP1 phosphorylation does not modulate this function. By means of partial proteolysis of purified recombinant VP1 capsomeres for assessing structural features of the protein domain affecting DNA binding, a trypsin-sensitive site at lysine 28 was found to eliminate VP1 binding to DNA. The binding constant of recombinant VP1 to polyomavirus DNA was determined by an immunoprecipitation assay (R. D. G. McKay, J. Mol. Biol. 145:471-488, 1981) to be 1 x 10(-11) to 2 x 10(-11) M, which was not significantly different from its affinity for plasmid DNA. McKay analysis of deleted VP1 proteins and VP1-beta-galactosidase fusion proteins indicated that the amino terminus was both necessary and sufficient for DNA binding. As shown by electron microscopy, DNA inhibited in vitro capsomere self-assembly into capsidlike structures (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea, Cell 46:895-904, 1986). Thus, VP1 is a high-affinity, non-sequence-specific DNA-binding protein with the binding function localized near its trypsin-accessible amino terminus. The inhibitory effects of disulfide reagents on DNA binding and of DNA on capsid assembly suggest possible intermediate steps in virion assembly.     

Virion assembly defect of polyomavirus hr-t mutants: underphosphorylation of major capsid protein VP1 before viral DNA encapsidation.

The major capsid protein of polyomavirus, VP1, was separated into at least four subspecies by isoelectric focusing. One of these subspecies was selectively extracted from purified virions by mild treatment with sodium dodecyl sulfate, leaving a 140S particle enriched in the other three forms. The two most acidic subspecies were labeled in vivo with [32P]phosphate, and these subspecies are among those identified as being deficient in nontransforming host range (hr-t) mutant virus nonpermissive infection of NIH3T3 cells. Quantitation of VP1 phosphorylation revealed that hr-t mutant virus VP1 is phosphorylated to about 40 to 50% the level of the wild type in NIH3T3 cells, and two-dimensional phosphoamino acid analysis suggested that threonine phosphorylation was affected more than serine phosphorylation. Two results indicate that the VP1 modifications occur before and independent of virus assembly: modified subspecies were detected during wild-type infection within a 2-min pulse-label with [32S]methionine, and VP1 modifications of temperature-sensitive VP1 mutants were the same at both restrictive and permissive temperatures for virus assembly. We conclude that most VP1 modification occurs before viral DNA encapsidation, and that one defect in hr-t mutant virus assembly is in VP1 phosphorylation, primarily affecting threonine.     

Polyomavirus major capsid protein VP1 is modified by tyrosine sulfuration.

Polyomavirus was propagated in primary mouse kidney cell monolayers and 35S-sulfate labeled by maintaining the infected cells in serum-free Eagle medium supplemented with 35S-labeled sodium sulfate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CsCI gradient-purified 35S-sulfate-labeled virions followed by fluorography indicated that the polyomavirus-coded major capsid protein VP1 incorporated this radiolabel. Two-dimensional gel electrophoresis followed by fluorography revealed 35S-sulfate incorporation into only two of the six VP1 isoelectric species (E and F). Amino acid analysis of 35S-sulfate labeled VP1 by enzymatic hydrolysis followed by two-dimensional thin-layer electrophoresis revealed the presence of 35S-sulfate-labeled tyrosine-O-sulfate.     

Identification of the threonine phosphorylation sites on the polyomavirus major capsid protein VP1: relationship to the activity of middle T antigen.

Phosphorylation of the polyomavirus major capsid protein VP1 was examined after in vivo 32P labeling of virus-infected cells. Two phosphorylated peptide fragments of VP1 were identified by protease digestion, high-performance liquid chromatography purification, mass spectrometry, and N-terminal sequencing. The peptides from residues 58 to 78 and residues 153 to 173 were phosphorylated on threonine. Site-directed mutations were introduced at these threonine sites, and mutant viruses were reconstructed. A threonine-to-glycine change at residue 63 (mutant G63) and a threonine-to-alanine change at residue 156 (mutant A156) resulted in viruses defective in phosphorylation of the respective peptides after in vivo labeling. Growth of the mutant G63 virus was similar to that of the wild-type virus, but the mutant A156 was inefficient in assembly of 240S viral particles. Polyomavirus nontransforming host range (hr-t) mutants are defective in VP1 threonine phosphorylation when grown in nonpermissive cells (R. L. Garcea, K. Ballmer-Hofer, and T. L. Benjamin, J. Virol. 54:311-316, 1985). Proteolytic mapping of VP1 peptides after in vivo labeling from hr-t mutant virus infections demonstrated that both residues T-63 and T-156 were affected. These results suggest that the block in virion assembly in hr-t mutant viruses is associated with a defect in phosphorylation of threonine 156.     

Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.     

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein

The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol μl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.     

Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.     

Production and characterization of monoclonal antibodies of shrimp White spot syndrome virus envelope protein VP28

BALB/c mice were immunized with purified White spot syndrome virus (WSSV). Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E. coli. in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish. Western-blot suggested that all these monoclonal antibodies were against the conformational structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling. These monoclonal antibodies could be used to develop immunological diagnosis methods for WSSV infection.     

Interactions of heterologous DNA with polyomavirus major structural protein, VP1

'Empty' polyomavirus pseudocapsids, self-assembled from the major structural protein VP1, bind DNA non-specifically and can deliver it into the nuclei of mammalian cells for expression [Forstová et al. (1995) Hum. Gene Ther. 6, 297-3061. Formation of suitable VP1-DNA complexes appears to be the limiting step in this route of gene delivery. Here, the character of VP1-DNA interactions has been studied in detail. Electron microscopy revealed that VP1 pseudocapsids can create in vitro at least two types of interactions with double-stranded DNA: (i) highly stable complexes, requiring free DNA ends, where the DNA is partially encapsidated; and, (ii) weaker interactions of pseudocapsids with internal parts of the DNA chain.     

Production of monoclonal antibodies to the prion protein and their characterization

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25–36 of PrP corresponds to one antibody, and epitopes located in region 222–229, to the other two. The antibodies to fragment 222–229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.