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PubMedDate not supplied

Visualization of three-dimensional density maps reconstructed from cryoelectron micrographs of viral capsids.

Full evaluation of three-dimensional density maps calculated from cryoelectron micrographs of complex supramolecular structures requires that the maps be sifted by a variety of complementary visualization techniques. We present here a primer for a number of such techniques in current widespread use, including surface rendering; serial sections; simulated motion; and real-time manipulation of tiled surfaces displayed on an advanced workstation. The principles on which these techniques operate are briefly reviewed, as are their advantages and limitations, with emphasis on the requirements for visual representation of viral capsid structures. These methods are illustrated in application to a density map of herpes simplex virus type 1 (HSV-1) capsid at 24 A resolution, which reveals more detailed information than heretofore concerning the inner surface of the icosahedral capsid shell and the 150-A-long channels that pass through each of the 162 capsomers.

Animals↗
PubMed2001

Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids.

The human parvovirus adeno-associated virus type 2 (AAV-2) possesses many features that make it an attractive vector for gene delivery in vivo. However, its broad host range may limit its usefulness and effectivity in several gene therapy applications in which transgene expression needs to be limited to a specific organ or cell type. In this study, we explored the possibility of directing recombinant AAV-2 transduction by incorporating targeting peptides previously isolated by in vivo phage display. Two putative loops within the AAV-2 capsid were examined as sites for incorporation of peptides. We tested the effects of deleting these loops and different strategies for the incorporation of several targeting peptides. The tumor-targeting sequence NGRAHA and a Myc epitope control were incorporated either as insertions or as replacements of the original capsid sequence. Viruses were assessed for packaging, accessibility of incorporated peptides, heparin binding, and transduction in a range of cell lines. Whereas recombinant viruses containing mutant capsid proteins were produced efficiently, transduction of several cell lines was significantly impaired for most modifications. However, certain mutants containing the peptide motif NGR, which binds CD13 (a receptor expressed in angiogenic vasculature and in many tumor cell lines), displayed an altered tropism toward cells expressing this receptor. Based on this work and previous studies, possible strategies for achieving in vivo targeting of recombinant AAV-2 are discussed.

Amino Acid Sequence↗
PubMed1998

Baculovirus-mediated large-scale expression and purification of a polyhistidine-tagged rubella virus capsid protein.

The capsid protein of rubella virus was produced in baculovirus-infected Spodoptera frugiperda insect cells, with a polyhistidine affinity tag at the carboxy terminus. The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal-ion affinity chromatography. Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. The final yield was 5 mg of purified protein per liter of cell culture. Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen-antibody interaction study. A specific interaction between the two proteins was shown. Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies.

Amino Acid Sequence↗
PubMed1999

Overexpression and purification of an immunologically reactive His-BIV capsid fusion protein.

The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)6 p26 capsid fusion protein. The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-beta-d-galactoside. Purification was based on interaction of the hexa-histidine polypeptide with metal ions. Expression could represent 11% of the total protein in Escherichia coli, allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture. The (His)6 p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein. The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples.

Animals↗
PubMed1993

Attachment of neutralizing antibodies stabilizes the capsid of poliovirus against uncoating.

Poliovirus capsid is rapidly and irreversibly degraded upon treatment in hypotonic buffer at 37 degrees C. However, the attachment of neutralizing antibodies stabilized the virus capsid against degradation by similar treatment. This was demonstrated by the use of antisera to poliovirus from rabbits and other mammalian species. As shown with neutralizing antibodies, the degree of capsid stabilization paralleled the number of bound IgG molecules per virion and the rate of neutralization. Upon acid treatment virus-antibody complexes disaggregated and infectious virus was recovered. Divalent binding of antibodies to the virus surface has been suggested to be a prerequisite for neutralization by cross-linking subunits, thereby preventing virus uncoating. This reaction was mimicked by using chemical cross-linking with a bifunctional reagent. The introduction of intramolecular bridges between adjacent VP1 and VP3 polypeptides was accompanied by a marked drop in infectivity. The treated virus was still able to adsorb to and penetrate into HeLa cells. Physically, intact virus could be reisolated from infected cells, suggesting that the loss of infectivity was probably due to the inhibition of uncoating. Additionally, cross-linked virus was stabilized against treatment in hypotonic buffer. Cleavage of the cross-links by treatment with a reducing agent under mild conditions resulted in restoration of the infectivity.

Animals↗
PubMed1993

The tobamovirus capsid protein functions as a host-specific determinant of long-distance movement.

The tobamoviruses, tobacco mosaic virus (TMV) and Odontoglossum ringspot virus (ORSV), differ in the range of plant species that each can systemically infect. Both viruses systemically infect Nicotiana benthamiana similarly, but differ in the ability to systemically infect tobacco (N. tabacum). ORSV is confined to the inoculated leaves of N. tabacum, whereas TMV causes a rapid systemic infection. Genetic chimeras were created to identify viral genes involved in specific interactions in long-distance movement in N. tabacum. In N. tabacum, a chimera expressing the ORSV 30-kDa protein exhibited a reduced rate of cell-to-cell spread, similar to that of ORSV, and, also like ORSV, was deficient in systemic invasion, implying a relationship between the rates of cell-to-cell and long-distance movement. However, a TMV chimera expressing the ORSV capsid protein gene spread cell to cell similarly to TMV, but was deficient in long-distance movement and systemic infection. These data suggest that tobamovirus capsid protein(s) may be required to interact with host components in a specific manner to allow efficient long-distance movement, and the ORSV capsid protein did not function in this manner in tobacco plants.

Base Sequence↗
PubMed1993

Baculovirus expression of gene 6 of the IDIR strain of group B rotavirus (GBR): coding assignment of the major inner capsid protein.

The polypeptide product of gene 6 of the IDIR strain of group B rotavirus was synthesized by means of a baculovirus expression system in order to confirm the coding assignment of the gene and to develop reagents broadly reactive with heterologous strains of Group B rotaviruses (GBR). Earlier experiments indicated that IDIR virus gene 6 encoded the group-specific, major inner capsid protein, but direct confirmation of this coding assignment was not previously reported. The expression of IDIR virus gene 6 from baculovirus recombinants resulted in production of a protein with an apparent molecular weight equivalent to that deduced from the gene sequence (44 kDa). In addition, larger recombinant proteins were also observed, and these appeared to be consistent in size with oligomers of the primary VP6 product. The expressed protein reacted with antibody directed against the IDIR agent and other strains of GBR, but no reaction was observed with antibody directed against group A rotavirus. Serologic reactivity was also observed between the gene 6 product and a monoclonal antibody directed against the GBR group-specific antigen. Antibody directed against the recombinant gene 6 product specifically reacted with the IDIR virus major inner capsid protein in an immunoblot format. These experiments conclusively demonstrated that IDIR virus gene 6 encoded the major inner capsid protein and confirmed the presence of group B-specific antigenic epitopes on the protein.

Animals↗
PubMed1993

HPV-1 capsids expressed in vitro detect human serum antibodies associated with foot warts.

Seventy-eight human serum samples were screened for their ability to immunoprecipitate the major (L1) and minor (L2) capsid proteins of HPV1. The L1 and L2 proteins expressed from a recombinant vaccinia virus were able to self assemble into capsids in the nuclei of infected cells. Twenty-eight of the sera precipitated the L1 protein. The L1 protein was only precipitated when the protein was native, denatured protein was not precipitated by the human sera. None of the sera precipitated the L2 protein. The assay demonstrated a significant association between the ability of sera to precipitate the L1 protein and a clinical history of foot warts (P = 0.001). The same serum samples were tested by immunoblots using L1 and L2-trpE bacterial fusion proteins. It was found that almost half of the sera reacted with the L2 fusion protein and few reacted with the L1 protein. Immunoblot results did not correlate well with a clinical history of foot warts (P = 0.7), suggesting that immune precipitation of capsid proteins may be superior to immunoblotting for serodiagnosis of HPV infections.

Adolescent↗
PubMed1993

Analysis of hepatitis C virus capsid, E1, and E2/NS1 proteins expressed in insect cells.

The baculovirus/insect cell expression system was used to express the capsid protein and glycoproteins (e1 and e2/NS1) of the hepatitis C virus (HCV). Each polypeptide domain was expressed individually using two different constructs varying at their carboxy termini in order to retain or delete hydrophobic domains that may be involved in membrane association. The capsid proteins were transported to the nucleus where they formed a single large crystal-like inclusion. The capsid proteins were phosphorylated in insect cells. The e1 and e2 polypeptides were present in both the soluble and insoluble cellular fractions. Deletion of a hydrophobic domain in the carboxy terminus of e2 resulted in the polypeptide becoming soluble but not secreted. Deletion of the carboxy terminus of e1 had no effect on solubility. Both e1 and e2 were glycosylated, with variable glycosylation of e1 giving rise to a series of polypeptides varying in apparent molecular weight. Co-infection of insect cells with viruses expressing e1 and e2 resulted in a complex that permitted the coimmunoprecipitation of e1 with antibodies to e2 and vice versa. Immunofluorescence staining of insect cells expressing e1 and e2 indicated that reactivity to e2 was more prevalent in anti-HCV positive human sera.

Animals↗
PubMed1994

Poliovirus type 3/Saukett: antigenic and structural correlates of sequence variation in the capsid proteins.

The Saukett/USA/50 strain is the type 3 component of the inactivated poliovirus vaccine. The capsid-coding region of genomic RNA of Saukett strains from five different sources was sequenced and the sequence differences were correlated with antigenic differences measurable with poliovirus type 3-specific neutralizing monoclonal antibodies. All strains appeared to have capsid protein genes identical in size to those of the entirely sequenced type 3 poliovirus strains. The nucleotide sequence identity between the strains was 91% on the average and the strains could be divided into three groups. Amino acid differences were seen in 30 positions located throughout the capsid region both within and outside the known antigenic sites. Substitutions at the known antigenic sites explained most of the observed antigenic differences. Use of the atomic coordinates of the crystal structure model of the Sabin 3 virus and prior data based on escape mutants and peptide scanning revealed that most of the exposed substitutions located outside the known antigenic sites are spatially associated with regions found to be antigenic by either or both of these methods.

Antigenic Variation↗
PubMed1994

Use of HPV 1 capsids produced by recombinant vaccinia viruses in an ELISA to detect serum antibodies in people with foot warts.

A sandwich ELISA was developed to detect HPV antibodies using HPV 1 capsids that were purified from recombinant vaccinia virus-infected cells and a monoclonal antibody to the HPV 1 L1 protein. Sera from 91 college-aged women who had been previously screened for HPV 1 antibodies by immune precipitation of capsid proteins were tested by ELISA. A cutoff point was established independently of other criteria based on the assumption that the ELISA values came from a mixture of two Normal distributions representing seropositive and seronegative individuals. It was found that the data fit this model best when the natural log of the ELISA (+0.5 to make all of the values positive) was used. Positive sera were shown to react with a conformational epitope(s) on the L1 protein. In the population reporting foot warts, 16 of 18 (89%) had ELISA values above the cutoff. This compared to 38 of 73 (53%) positives in the population reporting no history of foot warts. The odds ratio for the association of the ELISA reactivity with foot warts was 7.23 (95% CI 1.53, 69.4; P < 0.01). There was no significant association between the ELISA reactivity and wart infections reported at other sites. The average of the log ELISA values for individuals never reporting foot warts was -0.223 (SD 0.468), whereas the average value for individuals reporting foot warts within 10 years was 0.191 (SD 0.450) (P = 0.001). There was a negative correlation between the magnitude of ELISA reactivity and the time elapsed since the last appearance of foot warts. This apparent loss of seroreactivity over time may indicate that HPV 1 is usually eliminated from the host after infection or that inadequate levels of HPV 1 capsid antigen are produced during latent foot warts to maintain antibody levels.

Adolescent↗
PubMed1994

Mutations in an exposed domain of Sindbis virus capsid protein result in the production of noninfectious virions and morphological variants.

A critical event in the process of Sindbis virus assembly is the interaction of the virus nucleocapsid with the target membrane which has been modified by virus envelope glycoproteins. A specific association between the endodomain of the pE2/E2 glycoprotein and an unidentified domain in the capsid protein is responsible for this association. We have examined the nature of this association by the production of a mutant which has two amino acid substitutions in a domain of the capsid protein known to be exposed on the surface of the assembled nucleocapsid (Ser180/Gly183). These substitutions result in a mutant which is defective in production of infectious virus, especially at low (28 degrees) and high (39.5 degrees) temperatures. Although noninfectious, the mutant virus is as efficient at mediating cell-virus-cell fusion from without as is an equal amount of wild-type virus particles, suggesting that the envelope glycoproteins are functional and that the loss of infectivity may be due to an event after membrane fusion, possibly nucleocapsid uncoating. At low temperature but not high temperature, the mutant produces normal size virus particles (T = 4) and larger particles with a predicted triangulation number of 7. These data suggest that, at low temperature, the mutant capsid protein may assume two different conformations capable of assembling either T = 4 or T = 7 nucleocapsids.

Amino Acid Sequence↗
PubMed1994

Analysis of a type D retroviral capsid gene expressed in ovine pulmonary carcinoma and present in both affected and unaffected sheep genomes.

Ovine pulmonary carcinoma is a contagious neoplastic disease of sheep which can be transmitted experimentally with cell-free filtrate. Products homologous to structural proteins of type D and type B retroviruses are expressed in the tumors, but an infectious retrovirus has yet to be isolated. Retroviral RNA was isolated from an affected Peruvian sheep and used as a substrate for reverse transcription. The DNA obtained was subjected to polymerase chain reaction amplification using degenerate primers designed to amino acid sequences shared by the capsid peptides of the viruses that cross-react with the proteins found in the tumor tissues. A retroviral capsid region of type D lineage was isolated that proved to be highly homologous to the jaagsiekte sheep retrovirus isolated from affected sheep in South Africa. This capsid sequence was used to detect the virus in the genomes of sheep with and without ovine pulmonary carcinoma. More than 20 endogenous sequences that hybridized with the probe were found in the sheep examined, and assortment of these could be followed in sheep families. Initial experiments did not show any obvious differences in viral presence in the tumors compared with the background of endogenous viral sequences in unaffected lungs.

Amino Acid Sequence↗
PubMed1995

Interference with replication of two double-stranded RNA viruses by production of N-terminal fragments of capsid polypeptides.

It is possible to interfere with the replication of a number of plant RNA viruses by systemic production of viral capsid polypeptides or RNA-dependent RNA polymerases, or by production of untranslatable portions of viral plus strands or minus strands. Interference can occur by a number of mechanisms. We have discovered that the Saccharomyces cerevisiae double-stranded RNA viruses ScVL1 and ScVLa, which exist as permanent persistent infections of their host cells, can be cured very efficiently by production of N-terminal fragments of their capsid polypeptides. These totiviruses produce only two polypeptides: a capsid polypeptide (Cap) and a Cap-Pol fusion polypeptide with RNA-dependent RNA polymerase activity. Three types of interference can be detected: interference due to overproduction of both Cap and Cap-Pol, interference due to overproduction of Cap (and consequent distortion of the Cap to Cap-Pol ratio), and interference due to negative complementation by N-terminal fragments of Cap. Some N-terminal fragments of Cap appear to be incorporated into viral particles, but only in the presence of a complete Cap protein. We postulate that incorporation of N-terminal fragments of Cap results in the formation of defective particles.

Base Sequence↗
PubMed1996

The low pH-dependent entry of avian reovirus is accompanied by two specific cleavages of the major outer capsid protein mu 2C.

Avian reoviruses are capable of inducing rapid and extensive syncytium formation, a process that occurs preferentially under conditions of neutral or alkaline pH. In order to ascertain whether the membrane fusion-inducing capability of avian reovirus confers a pH-independent entry mechanism on the virus, virus entry was investigated using internalization assays and several lysomotropic agents that inhibit endosomal acidification. The ability of avian reovirus to infect cells was severely restricted under all conditions that prevented endosomal acidification. The decreased infection efficiency in the presence of the lysomotropic agents correlated with an inhibition in the proteolytic processing of the major outer capsid protein mu 2C. The importance, with respect to virus infection, of the low pH-dependent cleavage of the avian reovirus mu 2C protein was confirmed by demonstrating that infectious subviral particles, generated by proteolytic processing in vitro, were capable of efficiently infecting cells in the presence of the lysomotropic agents. These results indicated that avian reovirus entry-specific membrane interactions are largely dependent on an endosome-mediated proteolytic processing of the virus particle, suggesting that the syncytium-inducing properly of the sigma 3 protein is not sufficient to promote virus uptake. Furthermore, avian reovirus internalization was associated with two distinct cleavages of the major outer capsid protein mu 2C, unlike the entry-specific processing of the analagous mammalian reovirus major outer capsid protein mu 1C. The mu 2C cleavages occured sequentially and appeared to involve distinct cleavage specificities. Moreover, the second cleavage event was observed to be both virus strain- and cell type-independent, suggesting that the cleavage is both specific and biologically significant.

Animals↗
PubMed1996

Single amino acid changes in the murine leukemia virus capsid protein gene define the target of Fv1 resistance.

The mouse Fv1 genetic locus controls resistance to subgroups of ecotropic, MCF, and amphotropic murine leukemia viruses (MuLVs). In addition to the four previously defined alleles of Fv1 (Fv1(n), Fv1(b), Fv1(nr), Fv1(0)), we present evidence that the novel restriction pattern characteristic of DBA/2J mice maps to the Fv1 locus and therefore represents a novel allele, here designated Fv1(d). Previous studies had demonstrated that the Fv1-mediated viral tropism is determined within the capsid protein gene, and that N- and B-tropic virus capsids differ only in two adjacent amino acids. We introduced various amino acid substitutions at these two sites in the N-tropic AKV MLV capsid gene, and typed resulting viruses for host range on cells carrying all five Fv1 alleles as well as on cells from additional wild mouse species with Fv1-like differences in virus susceptibility. Results indicate that alteration of the first of the two amino acids does not alter tropism, but alteration of the second alone is sufficient to convert the N-tropic AKV MLV to a B-tropic virus. Substitution of leucine for arginine at this site produced a virus with an unusual tropism not characteristic of any of the naturally occurring or laboratory strains of MuLV.

Alleles↗
PubMed1997

Characterization of an overlapping CD8+ and CD4+ T-cell epitope on rubella capsid protein.

A synthetic peptide corresponding to rubella virus capsid protein residues 263 to 275 which contains an epitope recognized by a cloned CD4+ cytotoxic T-lymphocyte (CTL) line was used to induce CD8+ T-cell lines specific to this peptide. A peptide-specific CD8+ CTL clone was derived and characterized. This peptide-specific CD8+ CTL clone exhibited cytotoxicity against target cells infected by a vaccinia recombinant virus expressing rubella virus capsid protein, but not by target cells infected by vaccinia recombinant virus expressing rubella virus E1 or E2 envelope proteins. Analysis of HLA class I restriction of the CD8+ CTL clone revealed that A11 and A3 were restrictive elements. Fine mapping with truncated and overlapping peptide analogs revealed a nonamer sequence, C(264-272), as the T-cell epitope eliciting stronger cytotoxicity. Two anchor residues for binding to HLA A11 and A3 were identified at position 2 (isoleucine) and at position 9 (histidine) or at position 8 (arginine) of the epitope sequence. The identification of overlapping CD4+ and CD8+ T-cell epitopes within the capsid protein sequence C(263-275) implicates a strategy for using such epitopes in a candidate peptide-based rubella vaccine.

Amino Acid Sequence↗
PubMed1997

Crystals of Rous sarcoma virus capsid protein show a helical arrangement of protein subunits.

Crystals of Rous sarcoma virus (RSV) capsid protein diffract X rays to 3.5 A resolution and belong to the monoclinic space group C2 with unit cell parameters a = 374.4 A, b = 128.1 A, c = 200.2 A, and beta = 121.8 degrees. One asymmetric unit of the crystal may contain between 28 and 35 molecules, based on reasonable crystal density assumptions. A self-rotation function and Patterson synthesis suggest that RSV capsid protein crystallizes as a helical array. The determinants of the viral particle morphology are not encoded in the capsid alone. The assembly of a helical array in the crystal reflects the absence of any conformational switching. However, it is expected that the subunit interactions seen in the crystal will be preferred and will relate to those found in the immature or mature virion.

Amino Acid Sequence↗