Expression of enterovirus 70 capsid protein VP1 in Escherichia coli

The VP1 gene of enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5'-UGUCUCCUUUUC-3' flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21(DE3), Rosetta 2(DE3) or Rosetta (DE3)pLysS). High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli, high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli.     

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.     

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.     

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.     

Production and characterization of monoclonal antibodies to polyomavirus major capsid protein VP1.

Four hybridoma cell lines producing monoclonal antibodies against intact polyoma virions were produced and characterized. These antibodies were selected for their ability to react with polyoma virions in an enzyme-linked immunosorbent assay. The antibodies immunoprecipitated polyoma virions and specifically recognized the major capsid protein VP1 on an immunoblot. Distinct VP1 isoelectric species were immunoprecipitated from dissociated virion capsomere preparations. Two-dimensional gel electrophoresis demonstrated antibody reactivity with specific VP1 species. Monoclonal antibodies E7 and G9 recognized capsomeres containing VP1 species D, E, and F, while monoclonal antibodies C10 and D3 recognized capsomeres containing species B and C. Two of the monoclonal antibodies, E7 and G9, were capable of neutralizing viral infection and inhibiting hemagglutination. The biological activity of the monoclonal antibodies correlated well with the biological function of the species with which they reacted.     

The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.     

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.     

Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity

Enterovirus type 71 (EV71) causes hand, foot, and mouth disease (HFMD), which is mostly self-limited but may be complicated with a severe to fatal neurological syndrome in some children. Understanding the molecular basis of virus-host interactions might help clarify the largely unknown neuropathogenic mechanisms of EV71. In this study, we showed that human annexin II (Anx2) protein could bind to the EV71 virion via the capsid protein VP1. Either pretreatment of EV71 with soluble recombinant Anx2 or pretreatment of host cells with an anti-Anx2 antibody could result in reduced viral attachment to the cell surface and a reduction of the subsequent virus yield in vitro. HepG2 cells, which do not express Anx2, remained permissive to EV71 infection, though the virus yield was lower than that for a cognate lineage expressing Anx2. Stable transfection of plasmids expressing Anx2 protein into HepG2 cells (HepG2-Anx2 cells) could enhance EV71 infectivity, with an increased virus yield, especially at a low infective dose, and the enhanced infectivity could be reversed by pretreating HepG2-Anx2 cells with an anti-Anx2 antibody. The Anx2-interacting domain was mapped by yeast two-hybrid analysis to VP1 amino acids 40 to 100, a region different from the known receptor binding domain on the surface of the picornavirus virion. Our data suggest that binding of EV71 to Anx2 on the cell surface can enhance viral entry and infectivity, especially at a low infective dose.     

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.     

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.     

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.     

Simian virus 40 agnoprotein facilitates perinuclear-nuclear localization of VP1, the major capsid protein.

The agnoprotein of simian virus 40 (SV40) is a 61-amino-acid protein encoded in the leader of some late mRNAs. In indirect immunofluorescence studies with antisera against SV40 capsid proteins, we show that mutants which make no agnoprotein display abnormal perinuclear-nuclear localization of VP1, the major capsid protein, but not VP2 or VP3, the minor capsid proteins. In wild-type (WT) SV40-infected CV-1P cells, VP1 was found predominantly in the cytoplasm until 36 h postinfection (p.i.), approximately the time that high levels of agnoprotein became detectable under our infection conditions. Thereafter, VP1 localized rapidly to the perinuclear region and to the nucleus. In contrast, in agnoprotein-minus mutant-infected CV-1P cells, perinuclear-nuclear accumulation of VP1 occurred much less efficiently; a significantly greater fraction of cells with predominantly cytoplasmic fluorescence was observed up to 48 h p.i. At 48 and 60 h p.i., more cells with largely perinuclear and little nuclear staining were seen than in WT-infected controls. In similar analyses with stably transfected cell lines constitutively expressing the agnoprotein, VP1 localized to the nucleus before 30 h p.i., regardless of the infecting virus. Delayed nuclear entry of VP1 in a mutant which makes no agnoprotein was also overcome in a revertant which has a second site point mutation in VP1. This suggests that an alteration of VP1 can partially overcome the defect of the agnogene mutation by enhancement of the rate of its own nuclear localization. Taken together, these results indicate that at least one function of the agnoprotein is to enhance the efficiency of perinuclear-nuclear localization of VP1.     

Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells.

Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, alpha-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP70-P1 complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.     

High cooperativity of the SV40 major capsid protein VP1 in virus assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of approximately 6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.     

Mutational analysis of the carboxyl-terminal region of the SV40 major capsid protein VP1

Virus-like particles (VLPs), a promising next-generation drug delivery vehicle, can be formed in vitro using a recombinant viral capsid protein VP1 from SV40. Seventy-two VP1 pentamers interconnect to form the T = 7d lattice of SV40 capsids, through three types of C-terminal interactions, alpha-alpha'-alpha', beta-beta' and gamma-gamma. These appear to require VP1 conformational switch, which involve in particular the region from amino acids 301-312 (herein Region I). Here we show that progressive deletions from the C-terminus of VP1, up to 34 amino acids, cause size and shape variations in the resulting VLPs, including tubular formation, whereas deletions beyond 34 amino acids simply blocked VP1 self-assembly. Mutants carrying in Region I point mutations predicted to disrupt alpha-alpha'-alpha'-type and/or beta-beta'-type interactions formed small VLPs resembling T = 1 symmetry. Chimeric VP1, in which Region I of SV40 VP1 was substituted with the homologous region from VP1 of other polyomaviruses, assembled only into small VLPs. Together, our results show the importance of the integrity of VP1 C-terminal region and the specific amino acid sequences within Region I in the assembly of normal VLPs. By understanding how to alter VLP sizes and shapes contributes to the development of drug delivery systems using VLPs.     

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.     

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.     

Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein.

Treatment of purified polyoma virions with 6 M guanidine-hydrochloride and 0.01 M beta-mercaptoethanol resulted in the immediate loss of both hemagglutinating and plaque-forming ability. Gel filtration through Sepharose CL-6B beads allowed separation of the dimer, VP1, VP2, VP3, and histone proteins VP4-7 in highly purified form. Renaturation of the purified VP1 protein resulted in the formation of subunits that were morphologically, biophysically, and immunologically similar to native virion capsomeres.     

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.