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PubMed1990

Identification of the genes encoding two capsid proteins of herpes simplex virus type 1 by direct amino acid sequencing.

Amino-terminal amino acid sequencing was carried out on proteins purified from herpes simplex virus type 1 capsids. The sequences of two capsid proteins (VP19C and VP23) showed them to be encoded by genes UL38 and UL18, respectively. The product of UL38 has been shown to be essential for capsid assembly, but no role has previously been assigned to the product of UL18.

Amino Acid Sequence↗
PubMed1990

Synthesis of foot-and-mouth disease virus capsid proteins in insect cells using baculovirus expression vectors.

Foot-and-mouth disease virus (FMDV) cDNA cassettes containing sequences encoding the capsid precursor P1-2A with and without those encoding the proteases L and 3C were introduced into Autographa californica nuclear polyhedrosis virus (AcMNPV) expression vectors. Procapsid proteins 1AB, 1C and 1D were produced in cells infected with recombinant baculoviruses, when L and 3C were present in the constructs, indicating that these FMDV proteases were active in insect cells. Unlike P1 processing in poliovirus, which has been shown to be catalysed mainly by the 3CD gene product, the 3C protease of FMDV was able to process P1 independently of 3D. Cytotoxicity of the L protease for insect cells prevented the use of the optimized transfer vector, pAcRP23, for inserting L-containing cassettes into AcMNPV. By contrast, viable AcMNPV-FMDV recombinants could be made without restriction on choice of the transfer vector when the L gene was either not expressed or inactivated by an inframe deletion. In the latter case, normal cleavage at the L-P1 junction no longer occurred in cis, and a new processing event, probably catalysed by 3C, was observed within the C-terminal region of the residual L protein. Analysis of baculovirus-expressed products in sucrose gradients showed that a fraction of the capsid proteins is present in an aggregated form, migrating at 70S and possibly resembling FMDV empty capsid particles.

Animals↗
PubMed1990

Prokaryotic expression of the major capsid protein of human cytomegalovirus and antigenic cross-reactions with herpes simplex virus type 1.

The major capsid protein (MCP) of human cytomegalovirus (HCMV) was expressed in three portions as beta-galactosidase fusion proteins, covering about 75% of the open reading frame (ORF). Fusion protein SH 1 contained nucleotides 101 to 1243 of the ORF, fusion protein FS 1 contained nucleotides 1944 to 3089 and fusion protein SS 1 covered nucleotides 2624 to 3793. The recombinant proteins were tested for their immunoreactivity with human sera. Fusion protein FS 1 was found to represent the immunodominant region. The recombinant proteins were used to generate polyvalent rabbit antisera to investigate cross-reactivities with the major capsid protein (VP5) of herpes simplex virus type 1 (HSV-1). A monospecific antiserum raised against the fusion protein close to the N terminus of the MCP, as well as a monoclonal antibody and a monospecific rabbit antiserum directed against the viral MCP, cross-reacted with the VP5 as shown by immunoblotting and immunofluorescence. In order to detect common epitopes of the major capsid proteins of HCMV and HSV-1, the recombinant proteins were conjugated to CNBr-activated Sepharose and taken for purification of MCP antibodies from HCMV and HSV-1 seropositive individuals. Using this affinity chromatography method, cross-reactivity could be observed with HCMV- and HSV-positive human antisera in immunoblot experiments.

Antibodies, Monoclonal↗
PubMed1991

A temporal study of the expression of the capsid, cytoplasmic inclusion and nuclear inclusion proteins of tobacco etch potyvirus in infected plants.

Young leaves of tobacco, systemically infected by tobacco etch potyvirus (TEV), were examined for the presence and distribution of four virus encoded proteins [capsid, cytoplasmic inclusion (CI) and two nuclear inclusion (NI) proteins] at various time periods after inoculation of expanded leaves of the plants. The analyses were carried out by ELISA and by immunogold electron microscopy of thin sections of the leaves. All four proteins were detected simultaneously in the systemic leaves for the first time on the fifth day after inoculation of the expanded leaves. All four proteins increased in concentration until the seventh day and then showed no further increase with the exception of the capsid protein which continued to accumulate. The CI protein was first detected in association with the plasmalemma/cell wall and was subsequently found mostly in the form of pinwheels in the cytoplasm. The two NI proteins were found at all times after infection within the nucleus, although small concentrations were detected in the cytoplasm. These experiments suggest that both the NIa and NIb proteins are transported into the nucleus immediately after synthesis. At the earliest time periods after infection, high concentrations of these proteins (NIa and NIb) were found in their non-inclusion form in the nucleolus. At 14 days after infection, both proteins were found only as inclusions in the nucleus. The capsid protein was found at all stages of infection only in the cytoplasm.

Capsid↗
PubMed1992

Canine parvovirus empty capsids produced by expression in a baculovirus vector: use in analysis of viral properties and immunization of dogs.

The VP-2 genes of canine parvovirus (CPV) and a recombinant consisting of CPV and feline panleukopenia virus (FPV) sequences were cloned into baculovirus expression vectors, fused to the baculovirus polyhedrin promoter. Recombinant baculoviruses were prepared and the properties of the parvovirus proteins expressed in insect cells examined. The proteins produced were the same size as the authentic CPV VP-2 protein, and were produced late after infection; the quantity of proteins recovered from the insect cell cultures was similar to those produced in CPV infections. Parvovirus particles formed had the haemagglutination (HA), sedimentation and buoyant density properties of authentic CPV capsids. Both the CPV capsids and the CPV-FPV recombinant capsids from the baculovirus system expressed the same epitopes as those seen in the viable parvoviruses when tested with a panel of anti-parvovirus monoclonal antibodies. Lysates of recombinant baculovirus-infected cells were inoculated into dogs, giving rise to serum neutralizing and HA-inhibiting antibodies, and the immunized dogs were protected from clinical disease upon challenge with a virulent isolate of the most recent antigenic type of CPV.

Animals↗
PubMed1993

Herpes simplex virus type 1 capsid protein, VP21, originates within the UL26 open reading frame.

The goal of experiments reported here was to identify the genes that encode capsid proteins VP21 and VP24 of herpes simplex virus type 1 (HSV-1). Capsids were isolated from infected cells and the proteins were separated by SDS-PAGE. N-terminal amino acid sequence analysis of partial CNBr digestion products, and of intact VP21, showed that it is encoded within the UL26 open reading frame (ORF) of HSV-1 beginning with codon 248 and probably extending to the end of the ORF (codon 635). Similar analysis of digestion products confirmed that VP24 is specified by codons 1 to 247 at the 5' end of the UL26 ORF. Each of the seven known capsid proteins has now been assigned to an ORF.

Amino Acid Sequence↗
PubMed1997

Mapping of antigenic sites involved in neutralization on the capsid protein of feline calicivirus.

In order to locate amino acid residues involved in the formation of feline calicivirus (FCV) neutralizing epitopes, we analysed the capsid protein gene of monoclonal antibody neutralization-resistant variants of FCV. Amino acid substitutions in the variants were identified in the two hypervariable regions of the capsid protein. Four linear and two conformational epitopes were located in the regions from residues 426 to 460 and 490 to 520, respectively. The relative positions of individual epitopes agreed with our previous antigenic analysis. Two antigenic sites composed of the neutralizing epitopes were mapped in the hypervariable regions of the capsid protein, demonstrating that a relationship exists between the genetic variability and antigenic differences in the neutralization of FCV.

Animals↗
PubMed1998

Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus.

Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal antibodies (MAbs) and 12 overlapping fragments of the protein expressed in E. coli. Two antigenic regions were found. Ten out of the 11 MAbs recognized different discontinuous epitopes in the most immunodominant region of the viral capsid. This domain was located between residues 31 and 250 of the VP60 N terminus. The other MAb revealed the presence of an antigenic site within 102 aa of the C terminus. This MAb did not recognize the major cleavage product of the full-length 60 kDa protein. These results indicate that, in contrast to other caliciviruses such as Norwalk virus (NV), the 36 kDa cleavage product probably forms the N-terminal region of VP60. However, as in NV, the cleavage region appears to be the most immunodominant region.

Animals↗
PubMed1999

Structural similarities between influenza virus matrix protein M1 and human immunodeficiency virus matrix and capsid proteins: an evolutionary link between negative-stranded RNA viruses and retroviruses.

The evolutionary relationship of retroviruses to the negative-stranded RNA virus superfamily was examined by comparing protein structures. Since protein structures are more conserved over time than primary protein sequences, three-dimensional structural comparisons permit the identification of evolutionary relationships that were previously undetected. Human immunodeficiency virus (HIV) and influenza virus were used as representatives of the virus groups, and proteins with similar functions were compared. Since M1 of influenza virus has membrane- and RNA nucleocapsid-binding activities that are functionally analogous to those of the HIV matrix and capsid proteins, the structural similarities between these proteins were determined. Sequence alignments were based on superimposition of the three-dimensional structures. Helices 2, 2', 3 and 4 of the HIV matrix protein aligned and superimposed with the four-helix bundle of the membrane-binding N domain of M1 with a root mean square (RMS) of 3.48 A. Helices A, B and C of the HIV N-terminal capsid protein aligned and superimposed with three helices of the four-helix bundle of the RNA-binding N domain of M1 with an RMS of 2.63 A. The HIV Gag protein and influenza virus matrix protein may have evolved from a common ancestor protein. The similarities between influenza virus M1 and HIV matrix and capsid proteins may indicate an evolutionary link between retroviruses and negative-sense RNA viruses.

Amino Acid Sequence↗
PubMed2001

Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops.

Neutralization capsid epitopes are important determinants for antibody-mediated immune protection against papillomavirus (PV) infection and induced disease. Chimeric L1 major capsid proteins of the human PV type 16 (HPV-16) and the bovine PV type 1 (BPV-1) with a foreign peptide incorporated into several capsid surface loops self-assembled into pentamers or virus-like particles (VLP). Binding patterns of neutralizing monoclonal antibodies (MAb) and immunization of mice confirmed (i) that regions around aa 282-286 and 351-355 contribute to neutralization epitopes and identified the latter region as an immunodominant site and (ii) that placing a foreign peptide in the context of an assembled structure markedly enhanced its immunogenicity. Pentamers disassembled from wild-type HPV-16 and BPV-1 VLPs displayed some of the neutralization epitopes that were detected on fully assembled VLPs, but were deficient for binding a subset of neutralizing MAb that inhibit cell attachment.

Animals↗
PubMed2001

Comparison of capsid sequences from human and animal astroviruses.

We have sequenced the genomic 3'-end, including the structural gene, of human astrovirus (HAstV) serotype 7 and morphologically related viruses infecting pig (PAstV), sheep (OAstV) and turkey (TAstV-1). These sequences were compared with corresponding astrovirus sequences available in the nucleic acid databases, including sequences of the seven other HAstV serotypes, two other avian astroviruses (TAstV-2 and avian nephritis virus) and astrovirus from cat (FAstV). A 35 nt stem-loop motif near the 3'-end of the genome, previously described as being highly conserved, was present in all of the astroviruses except TAstV-2. In the N-terminal half of the capsid precursor protein, there were several short conserved peptide motifs. Otherwise the capsid proteins of astroviruses infecting different hosts were highly divergent. Calculation of genetic distances revealed that the distance between FAstV and HAstV is comparable to the largest distances between different HAstV serotypes. Higher similarities between the HAstV, FAstV and PAstV capsid sequences suggest interspecies transmissions involving humans, cats and pigs relatively recently in the evolutionary history of astroviruses.

3' Untranslated Regions↗
PubMed2002

The first hydrophobic domain of the hepatitis C virus E1 protein is important for interaction with the capsid protein.

The interaction between the hepatitis C virus capsid protein and the envelope protein E1 has been demonstrated previously in vivo. To determine the binding region of the E1 protein with the capsid protein, this interaction was characterized in vitro. This study shows that the interaction between these proteins should occur in the endoplasmic reticulum membrane rather than in the cytosol and that the first hydrophobic domain of the E1 protein (aa 261-291) is important for the interaction with the capsid protein.

Binding Sites↗
PubMed2003

Characterization of a recombinant type 3/type 2 poliovirus isolated from a healthy vaccinee and containing a chimeric capsid protein VP1.

A Sabin 3/Sabin 2/Sabin 3 (S3/2/3) intertypic recombinant poliovirus was isolated from a faecal specimen from a 2-year-old healthy boy approximately 12 weeks after administration of oral poliovirus vaccine. The first recombination junction was in the genomic region encoding the VP1 capsid protein between nucleotide positions 3274 and 3285 (numbering according to Sabin 3) and the second was in the RNA polymerase region (nucleotide positions 6824 and 6825). The recombination had introduced six Sabin 2-derived amino acids into the Sabin 3 capsid environment in the carboxyl terminus of VP1. The complete genome of the recombinant virus differed from corresponding parental Sabin strains at 33 nucleotide positions, nine of them resulting in an amino acid substitution. Four substitutions were in the capsid proteins and five were in the region encoding the non-structural proteins. One amino acid was changed in the antigenic site 2B and two in site 3B. In addition, the whole antigenic site 3A was replaced by Sabin 2-specific amino acids, but the antigenic characteristics of the S3/2/3 did not show type 2-specific features. Neutralizing antibody titres in sera from Finnish children immunized with the inactivated poliovirus vaccine were not lower against the recombinant virus than against Sabin 3. Our results suggest that the chimeric virus was most likely generated by recombination events in the vaccinee, rather than representing progeny of circulating vaccine-derived virus.

Amino Acid Substitution↗
PubMed2003

Model of the equine rhinitis A virus capsid: identification of a major neutralizing immunogenic site.

Mouse monoclonal antibodies (mAbs) were employed to select neutralization escape mutants of equine rhinitis A virus (ERAV). Amino acid changes in the ERAV mutants resulting in resistance to neutralization were identified in capsid protein VP1 at Lys-114, Pro-240 and Thr-241. Although the changes were located in different parts of the polypeptide chain, these mutants exhibited cross-resistance against all four mAbs employed, indicating that these residues contribute to a single immunogenic site. To explain this result, we constructed a model of the three-dimensional structure of the ERAV capsid based on comparison with the closely related foot-and-mouth disease virus (FMDV O(1)). According to this model, VP1 is folded so that Lys-114 is in the beta E-beta F loop of the polypeptide chain at a considerable distance from Pro-240 and Trp-241 in the C-terminal region. However, around the fivefold axis of symmetry, the C terminus of VP1 in each protomer extends to the beta E-beta F loop of the adjacent VP1 in the next protomer. We therefore propose that the immunogenic site in ERAV is formed as a result of the close proximity of the Lys-114 residue in the beta E-beta F loop of one VP1 molecule and of the Pro-240/Thr-241 residues in the adjacent VP1 polypeptide chain. In terms of the overall architecture of the viral capsid structure, this site in ERAV most closely resembles the immunogenic site 1 of FMDV O(1).

Amino Acid Sequence↗
PubMed2004

Selection pressures in the capsid genes of plant RNA viruses reflect mode of transmission.

To determine the selection pressures faced by RNA viruses of plants, patterns of nonsynonymous (dN) and synonymous (dS) substitution in the capsid genes of 36 viruses with differing modes of transmission were analysed. This analysis provided strong evidence that the capsid proteins of vector-borne plant viruses are subject to greater purifying selection on amino acid change than those viruses transmitted by other routes and that virus-vector interactions impose greater selective constraints than those between virus and plant host. This could be explained by specific interactions between capsid proteins and cellular receptors in the insect vectors that are necessary for successful transmission. However, contrary to initial expectations based on phylogenetic relatedness, vector-borne plant viruses are subject to weaker selective constraints than vector-borne animal viruses. The results suggest that the greater complexity involved in the transmission of circulative animal viruses compared with non-circulative plant viruses results in more intense purifying selection.

Animals↗
PubMed2007

Capsid stability and replication of human immunodeficiency virus type 1 are influenced critically by charge and size of Gag residue 183.

Structural data support a model where - following proteolytic cleavage--the amino-terminal domain of human immunodeficiency virus type 1 (HIV-1) capsid protein refolds into a beta-hairpin/helix tertiary structure that is stabilized by a buried salt bridge forming between the positively charged primary imino group of a proline residue and the negatively charged carboxyl group of a conserved aspartate. In order to evaluate the contribution of either side-chain length or charge to the formation of infectious virus capsids, aspartate 183 was substituted for glutamate or asparagine in the viral context. It was found that both modifications abolished infectivity of the corresponding viruses in permissive T lymphocytes, although none of particle assembly and release, RNA encapsidation, incorporation of Env glycoproteins and packaging of cyclophilin A were impaired. However, whereas biophysical analyses of mutant virions yielded wild-type-like particle sizes and densities, electron microscopy revealed aberrant core morphologies that could be attributed to either increased (D183N) or reduced (D183E) capsid stability. Although the two amino acid substitutions had opposing effects upon core stability, both mutants were shown to exhibit a severe block in early reverse transcription, underscoring the importance of correct salt-bridge formation for early steps of virus replication.

Amino Acid Substitution↗
PubMed2007

Reduced redox potential of the cytosol is important for African swine fever virus capsid assembly and maturation.

Assembly of African swine fever virus (ASFV) involves the transfer of the major capsid protein, p73, from the cytosol onto the cytoplasmic face of endoplasmic reticulum-derived membranes. During this process, the folding of p73 is dependent upon transient association with a specific viral chaperone, CAP80. The cell cytoplasm maintains high concentrations of reduced glutathione, leading to a reducing environment. Here, the effects of redox environment on the assembly of ASFV have been studied. Diamide, which oxidizes the cell cytosol, slowed the folding of p73 and prevented release from CAP80 and subsequent binding of p73 to membranes. Similarly, cell oxidation slowed the assembly of p73 molecules already bound to membranes into virus capsid precursors. Interestingly, addition of oxidized glutathione to newly assembled virus capsid precursors in vitro led to disassembly; however, virus particles released from cells were resistant to oxidized glutathione. These data show that assembly of ASFV requires the reducing environment that prevails in the cytosol, but as the virus matures, it becomes resistant to oxidation, possibly indicating preparation for release from the cell.

African Swine Fever Virus↗
PubMed2003

Crystallization and preliminary analysis of a dsDNA bacteriophage capsid intermediate: Prohead II of HK97.

HK97 Prohead II is an early intermediate in the maturation of HK97, a T = 7 dsDNA-tailed bacteriophage related to bacteriophage lambda. Previously, selected capsid-protein genes of HK97 were expressed in Escherichia coli and spontaneously assembled to form an icosahedral capsid that followed a maturation pathway closely similar to the authentic virion. The crystal structure of the mature HK97 capsid (Head II) made in this way was reported at 3.5 A resolution. Additional high-resolution structures of intermediates are needed to understand the maturation mechanism. The crystal structure of expressed Prohead II will elucidate the early steps of HK97 assembly. Crystals of the Prohead II mutant W336F were grown in 0.1 M HEPES pH 7.5, 0.2 M CaCl(2) and 2-3% PEG 4000 at a Prohead II concentration of 16.5 mg ml(-1). It was not possible to grow high-quality crystals of wild-type Prohead II. Diffraction was observed to 5 A resolution from these crystals on beamline 14BM-C at the Advanced Photon Source and data were collected to 5.5 A with a completeness of 77%. The space group was P2(1)3, with unit-cell parameter a = 707.0 A and four particles in the unit cell. The particles are on the body diagonals of the cubic cell, with icosahedral threefold axes coincident with crystallographic threefold axes. Self-rotation function and locked-rotation function analysis determined the particle orientation and a one-dimensional R-factor search along the body diagonal indicated that the particle centers were close to (1/4, 1/4, 1/4) and symmetry-related positions. Molecular-replacement averaging and phase extension are under way.

Bacteriophages↗