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PubMed2006

A free energy cascade with locks drives assembly and maturation of bacteriophage HK97 capsid.

We investigated the thermodynamic basis of HK97 assembly by scanning calorimetry and cryo-electron microscopy. This pathway involves self-assembly of hexamers and pentamers of the precursor capsid protein gp5 into procapsids; proteolysis of their N-terminal Delta-domains; expansion, a major conformational change; and covalent crosslinking. The thermal denaturation parameters convey the changes in stability at successive steps in assembly, and afford estimates of the corresponding changes in free energy. The procapsid represents a kinetically accessible local minimum of free energy. In maturation, it progresses to lower minima in a cascade punctuated by irreversible processes ("locks"), i.e. proteolysis and crosslinking, that lower kinetic barriers and prevent regression. We infer that Delta-domains not only guide assembly but also restrain the procapsid from premature expansion; their removal by proteolysis is conducive to initiating expansion and to its proceeding to completion. We also analyzed the mutant E219K, whose capsomers reassemble in vitro into procapsids with vacant vertices called "whiffleballs". E219K assemblies all have markedly reduced stability compared to wild-type gp5 (DeltaT(p) approximately -7 degrees C to -10 degrees C; where T(p) is the denaturation temperature). As the mutated residue is buried in the core of gp5, we attribute the observed reduction in stability to steric and electrostatic perturbations of the packing of side-chains in the subunit interior. To explain the whiffleball phenotype, we suggest that these effects propagate to the capsomer periphery in such a way as to differentially affect the stability or solubility of dissociated pentamers, leaving only hexamers to reassemble.

Bacteriophages↗
PubMed2006

A thermally induced phase transition in a viral capsid transforms the hexamers, leaving the pentamers unchanged.

Scanning calorimetry combined with cryo-electron microscopy affords a powerful approach to investigating hierarchical interactions in multi-protein complexes. Calorimetry can detect the temperatures at which certain interactions are disrupted and cryo-EM can reveal the accompanying structural changes. The procapsid of bacteriophage HK97 (Prohead I) is a 450A-diameter shell composed of 60 hexamers and 12 pentamers of gp5, organized with icosahedral symmetry. Gp5 consists of the N-terminal Delta-domain (11kDa) and gp5* (31 kDa): gp5* forms the contiguous shell from which clusters of Delta-domains extend inwards. At neutral pH, Prohead I exhibits an endothermic transition at 53 degrees C with an enthalpy change of 14 kcal/mole (of gp5 monomer). We show that this transition is reversible. To capture its structural expression, we incubated Prohead I at 60 degrees C followed by rapid freezing and, by cryo-EM, observed a capsid species 10% larger than Prohead I. At 11A resolution, visible changes are confined to the gp5 hexamers. Their Delta-domain clusters have disappeared and are presumably disordered, either by unfolding or dispersal. The gp5* hexamer rings are thinned and flattened as they assume the conformation observed in Expansion Intermediate I, a transition state of the normal, proteolysis-induced, maturation pathway. We infer that, at ambient temperatures, the hexamer Delta-domains restrain their gp5* rings from switching to a lower free energy, EI-I-like, state; above 53 degrees, this restraint is overcome. Pentamers, on the other hand, are more stably anchored and resist this thermal perturbation.

Bacteriophages↗
PubMed2004

Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses.

Noroviruses (NoV), previously called "Norwalk-like viruses", have emerged as the single most important cause of acute gastroenteritis worldwide. Most diagnostic reverse transcription-polymerase chain reaction (RT-PCR) assays target the viral RNA-dependent RNA polymerase; however, the major capsid protein (VP1) is the reference genomic region for establishing genotypes. In this study, we analyzed complete NoV VP1 sequences (n=100) and determined a region (region D) that was most suitable to differentiate between genotypes. Within region D, we designed two genogroup specific, broadly reactive, degenerate primer sets (GI and GII). The region D primers were evaluated in a single-tube one-step RT-PCR assay using a panel of 81 (31 GI, 50 GII) NoV strains from both outbreaks and sporadic cases. In total, 95% of the samples tested positive using the new region D primer sets. Phylogenetic analysis of region D sequences (36 deduced amino acids for GI, 56 deduced amino acids for GII), revealed 19 clusters (7 within GI and 12 within GII) including three new genetically distinct clusters, two of which were unresolved using region A sequences. Phylogenetic analysis of the complete VP1 sequences revealed identical grouping of strains and confirmed the newly identified clusters using region D. In summary, we successfully developed and evaluated a broadly reactive RT-PCR assay for reliable genotyping of GI and GII noroviruses.

Base Sequence↗
PubMed2007

Binding and neutralization efficiencies of monoclonal antibodies, Fab fragments, and scFv specific for L1 epitopes on the capsid of infectious HPV particles.

We compared the neutralization abilities of individual monoclonal antibodies (MAb) of two large panels reactive with L1 epitopes of HPV-11 or HPV-16. Binding titers were compared using both L1-only VLPs and L1/L2 pseudovirions. While the VLPs were antigenically similar to the pseudovirions, clear differences in the surface exposure of some epitopes were evident with the HPV-16 particles. To determine whether all antibody binding events are equivalent in their neutralizing effect on infectious HPV virions or pseudovirions, the binding and neutralization titers for individual MAbs were used to calculate the relative neutralization efficiency for each antibody. HPV neutralization was achieved by all MAbs capable of strong binding to either linear or conformation-sensitive epitopes on pseudovirus particles. Our data suggest, however, that some L1 epitopes may be more neutralization-sensitive than other surface epitopes, in that successful infection can be blocked by varying degrees of epitope saturation. Additionally, the effective neutralization of virions by several monovalent Fab fragments and single-chain variable fragments (scFv) demonstrates that viral neutralization does not require HPV particle aggregation or L1 crosslinking. Identification of capsid protein structures rich in neutralization-sensitive epitopes may aid in the development of improved recombinant vaccines capable of eliciting effective and long-term antibody-mediated protection against multiple HPV types.

Antibodies, Monoclonal↗
PubMed2005

Altering AAV tropism with mosaic viral capsids.

Over the past decade, AAV-based vectors have emerged as promising candidates for gene therapeutic applications. Despite the broad tropism of the first eight serotypes identified, certain cell types are refractory to transduction with AAV-based vectors. Furthermore, for certain applications the targeting of specific cell types is desirable. To improve on present methods to alter AAV2 tropism, we take advantage of AAV2 mosaics. Here, we show that AAV2 mosaics have improved infectivity compared with all-mutant virions. Using an AAV2 mutant that contains the immunoglobulin-binding Z34C fragment of protein A, we demonstrate the utility of AAV2 mosaics to alter AAV2 tropism. This system allows us to transduce selectively and efficiently MO7e and Jurkat cells. The use of AAV2 mosaics with a protein A fragment inserted into their capsid, together with targeting antibodies, is a versatile method that allows the specific transduction of a wide array of cell types.

Capsid↗
PubMed2000

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.

Animals↗
PubMed2003

Identification of a nonconventional motif necessary for the nuclear import of the human parvovirus B19 major capsid protein (VP2).

Human parvovirus B19 replicates and encapsidates its genome in the nucleus of erythroid progenitors in vivo and in vitro. We wanted to understand the determinants necessary for the nuclear transport of the major coat protein, VP2, which makes up about 96% of the viral capsid proteins. A nonconsensus basic motif, KLGPRKATGRW, necessary for the nuclear localization of VP2 was identified and shown to be able to import reporter proteins into the nucleus. The sequence is conserved among the VP2 C-terminal region of erythroviruses. This newly identified sequence will facilitate the understanding of the replication of these viruses.

Amino Acid Motifs↗
PubMed1993

Glycosylation of the major outer capsid protein of bluetongue viruses.

The major outer capsid protein, VP5, of five US bluetongue viruses, was found to be glycosylated. Enzymatic removal of the carbohydrate moiety did not affect the electrophoretic mobility of VP5 in SDS-PAGE, indicating the presence of only short and possibly unbranched oligosaccharide chains. The surface accessibilities and immunogenic specificities of two conformational-dependent antigenic epitopes on VP5 of BTV were not affected by deglycosylation. Selective binding of those lectins which have well-defined sugar specificities suggests that the potential short N-linked carbohydrate chain present on BTV VP5 might be composed of sialic acid alpha(2-6)-N-acetylgalactosamine-beta(1-3)-galactose-beta(1-4)-N- acetylglucosamine-beta(1-2)-mannose-N-acetylglucosamine-Asn. In accordance with the standard nomenclature, BTV-VP5 should now be termed GP5 due to the presence of the carbohydrate moiety.

Antibodies, Monoclonal↗
PubMed1998

Stability of polyomavirus major capsid protein VP1 under denaturants guanidine hydrochloride and urea.

The guanidine hydrochloride (GdnHCl)- and urea-induced equilibrium denaturation of recombinant polyomavirus (Py) major capsid protein VP1 was studied by circular dichroism and fluorescence spectroscopy. Both secondary and tertiary structures of PyVP1 were shown to be disrupted in the presence of denaturants. Although the far-UV circular dichroism (CD) spectra of PyVP1 in the denaturants exhibit similar two-phase transition as those obtained from the fluorescence measurements, the unfolding of PyVP1 in GdnHCl was shown to be more complex than a similar two-state mechanism. The presence of unfolding intermediates is manifested by the noncoincidence of transitions when detected by different probes. The unfolding intermediate appeared to be stabilized by 1 M NaCl. Addition of Ca2+ and 2-mercaptoethanol does not show significant effect on the conformational stability of PyVP1. Unfolding of PyVP1 in GdnHCl was shown to be an irreversible process.

Capsid↗
PubMed2001

Type-independent detection of foot-and-mouth disease virus by monoclonal antibodies that bind to amino-terminal residues of capsid protein VP2.

The characterization of monoclonal antibodies raised against the foot-and-mouth disease virus isolates A22 Iraq/1964, Asia1 Shamir-Israel/1989, and SAT1 Zimbabwe/1989 with regard to neutralizing activity and sensitivity of their epitopes for treatment with trypsin, resulted in the identification of one non-neutralizing antibody in each panel that binds to a trypsin-sensitive epitope. Furthermore, each of these antibodies recognized 27 isolates of different provenance, representative of six serotypes. These antibodies are recommended for type-independent antigen detection by ELISA. The epitopes for these antibodies reside at the intertypically conserved N-terminus of capsid protein VP2. The two are specified by the lysines at positions two and three, but differ from each other as indicated by the variable heavy chain sequences of their antibodies.

Amino Acid Sequence↗
PubMed2002

Expression and antigenic characterization of the major capsid proteins of human polyomaviruses BK and JC in Saccharomyces cerevisiae.

BK and JC viruses are ubiquitous human polyomaviruses that are associated with post-transplant interstitial nephritis (BK virus) and progressive multifocal leucoencephalopathy (JC virus). The use of a yeast system to express the major capsid protein (VP1) of two antigenic variants of BKV (strains SB and AS) and JCV is described. VP1s of AS and JCV expressed in Saccharomyces cerevisiae produced proteins of expected molecular weight as determined by gel electrophoresis whereas that of SB appeared to be lower than anticipated. However, all VP1s self-assembled into virus-like particles (VLP) retaining sialic acid-binding and antigenic properties of native virions. This method is highly efficient for producing recombinant proteins and therefore provides an alternative to the baculovirus system.

Antigens, Viral↗
PubMed2002

Differentiation of type A, Asia1 and O foot-and-mouth disease virus variants, amplified by the same system, by sequencing of the capsid protein genes.

A reverse transcription-dependent polymerase chain reaction (RT-PCR) is described that amplifies the genes encoding the capsid proteins VP1-3 of at least three evolutionary lineages each of the foot-and-mouth disease (FMD) virus types A, Asia1 and O. Most of these lineages are circulating at present in Asia and Africa. The method is not only suitable to confirm suspected outbreaks of FMD, but also describes the modulation of major and minor antigenic sites in the course of an epizootic by nucleotide sequence determination of the obtained RT-PCR products. Such knowledge helps to choose suitable vaccines for disease control.

Animals↗
PubMed2000

Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies.

Six neutralizing monoclonal antibodies (Mabs) and nine neutralization resistant viral variants (escape-mutant viruses (EMVs)) were used to further characterize the neutralization determinants of bluetongue virus serotype 10 (BTV10). The EMVs were produced by sequential passage of a highly cell culture adapted United States prototype strain of BTV10 in the presence of individual neutralizing Mabs. Mabs were characterized by neutralization and immune precipitation assays, and phenotypic properties of EMVs were characterized by neutralization assay. Sequencing of the gene segments encoding outer capsid proteins VP2 and VP5 identified mutations responsible for the altered phenotypic properties exhibited by individual EMVs. Amino acid substitutions in VP2 were responsible for neutralization resistance in most EMVs, whereas an amino acid substitution in VP5, without any change in VP2, was responsible for the neutralization resistance of one EMV. The data confirm that VP2 contains the major neutralization determinants of BTV, and that VP5 also can influence neutralization of the virus. The considerable plasticity of the neutralization determinants of BTV has significant implications for future development of non-replicating vaccines.

Amino Acid Substitution↗
PubMed2003

Protective immune response of the capsid precursor polypeptide (P1) of foot and mouth disease virus type 'O' produced in Pichia pastoris.

Foot and mouth disease virus (FMDV) is the aetiological agent of a highly contagious vesicular disease of cloven-hooved animals. The gene coding for the capsid polyprotein (P1) of FMDV from serotype 'O' vaccine strain (O75Madras) was cloned and expressed in yeast Pichia pastoris. The expressed P1 protein was characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot analysis. Immunisation of Guinea pigs with recombinant P1 induced FMDV type O specific immune response. The humoral response to vaccine was measured by indirect ELISA and a serum neutralisation test (SNT). The Guinea pig sera showed high titres both in ELISA and SNT. Upon challenge with virulent Guinea pig adapted homologous type 'O' virus, the animals showed a protective index of 2.52. This study shows that the yeast expressed FMDV P1 could be a safe vaccine in non-endemic countries and a cost-effective vaccine in endemic countries. This is the first report on the production of FMDV structural proteins in yeast and their application as a vaccine.

Animals↗
PubMed2002

Early protection against homologous challenge after a single dose of replication-defective human adenovirus type 5 expressing capsid proteins of foot-and-mouth disease virus (FMDV) strain A24.

Previously we demonstrated that two doses of a replication-defective human adenovirus serotype 5 (Ad5) carrying the capsid (P1) and 3C protease coding regions of a laboratory strain of FMDV (A12) completely protected five of six swine challenged with homologous virus. The objective of the current study was to evaluate the efficacy of one dose of an Ad5-vectored vaccine expressing the P1 coding region of an FMDV field strain. A replication-defective Ad5 containing the P1 coding region of FMDV A24 and the 3C coding region of A12 (Ad5A24) was constructed and evaluated for its ability to induce neutralizing antibodies and protect swine against homologous challenge after a single vaccination. Animals were challenged 7, 14 or 42 days after vaccination. Control groups included animals inoculated with commercial vaccine or phosphate-buffered saline. All vaccinated swine were completely protected against homologous challenge at 7, 14 or 42 days after vaccination. Based on these results, we conclude that a single inoculation of Ad5-vectored vaccines could be used as a tool to control FMD in outbreak situations.

Adenoviruses, Human↗
PubMed2002

Analysis of the capsid protein gene of a feline-like calicivirus isolated from a dog.

The authors report the sequence analysis of the capsid protein-encoding gene (ORF2) of a calicivirus strain recently isolated in Italy from a pup with enteritis. Sequence comparison and phylogenetic analysis revealed that the isolate is highly similar to field and reference feline caliciviruses (FCVs). The isolation of a feline-like calicivirus and the results of recent serological investigations suggest that FCV infection frequently occurs in dogs, but the consequences of this interspecies infection in the canine host are still unknown.

Amino Acid Sequence↗
PubMed1998

The cauliflower mosaic virus capsid protein: assembly and nucleic acid binding in vitro.

The capsid protein of the cauliflower mosaic virus (CaMV) was expressed in a bacterial system to study CaMV assembly. Bacterial lysates contained soluble particulate material and insoluble inclusion bodies that were both used for analysis. In vitro renaturation of pIV derivatives lead to the appearance of folded sheets or large tubular structures in electron microscopy. The region between amino acid positions 77 and 332 is sufficient for self-aggregation of pIV in vitro. C-terminal deletion to amino acid position 265 still allowed dimerization but prevented further aggregation. Nucleic acid binding assays of immobilized pIV derivatives demonstrated that a region located upstream of the retroviral "zinc finger-like" motif is involved in unspecific binding dsDNA, ssDNA and RNA.

Amino Acid Sequence↗
PubMed1998

Circular dichroism and fluorescence studies of polyomavirus major capsid protein VP1.

The conformational changes of polymavirus (Py) major capsid protein VP1 in solution by the solution pH, addition of calcium, and ionic strength were examined by circular dichroism (CD) and fluorescence spectroscopy. Comparison of the predicted secondary structures of PyVP1 and simian virus (SV) 40 by the methods of Chou-Fasman, Garnier et al., and Yang method are presented. Hydropathicity, surface probability, and chain flexibility of PyVP1 were computer-analyzed by the methods of Kyte and Doolittle, Emini et al., and Karplus and Schulz, respectively. The CD measurements indicate that the secondary structure of PyVP1 is little dependent on its concentration, Ca2+ concentration, and ionic strength, but is strongly pH dependent. Fluorescence studies showed that emission spectra of PyVP1 are also pH-dependent. At extreme acidic and alkaline pH, the fluorescence intensity of PyVP1 is decreased and the emission maximum is red-shifted. The fluorescence of PyVP1 is quenched by the presence of CsCl, KI, and acrylamide. The analyses of the modified Stern-Volmer plots indicate that five of seven tryptophan residues in PyVP1 are located on the surface of the protein, among which two are accessible to Cs+ and the other three are accessible to I- . The two others are buried more deeply in the interior of the protein molecule.

Acrylamide↗