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PubMed1979

Cell-free assembly of a polyoma-like particle from empty capsids and DNA.

A polyoma-like particle (PLP) is formed when polyoma DNA and purified empty capsids are incubated in a cell-free system. The DNA of this new particle is protected against the action of pancreatic DNase. The density of the purified PLP in CsCl is 1.32 g/cm3, which is intermediate between that of polyoma virions (1.34 g/cm3) and empty capsids (1.29 g/cm3). Purified PLP sediments at 190 S in sucrose and is stable in solutions of high ionic strength. When the DNA is extracted from PLP by the use of detergent and phenol, it is found to be doublestranded with a molecular weight of approximately 1.1 x 10(6). The particles are stable in CsCl at 4 degrees for at least 5 months. Electron micrographs indicate that highly purified PLPs stained with 2% PTA have the same appearance as polyoma capsids. Neither aggregates nor complexes bound by loose ionic bonds appear reasonable to explain these results. The evidence indicates that the DNA of this new polyoma-like particle, made under cell-free conditions, is protected by the capsid.

Cell-Free System↗
PubMed1996

Induction of SIV capsid-specific CTL and mucosal sIgA in mice immunized with a recombinant S. typhimurium aroA mutant.

We have developed a new expression system based on the E. coli groEL promoter. The suicide vector constructed (called APC vector) allows simultaneous attenuation of a Salmonella strain by disruption of the coding sequence for aroA and stable integration of a gene into the bacterial chromosome. High-level expression of antigen is achieved after Salmonella is taken up by macrophages, a major antigen processing cell of the host. The chloramphenicol acetyltransferase (CAT) and the simian immunodeficiency virus capsid (p27gag) genes were cloned downstream of the groEL promoter and expressed within S. typhimurium. By measuring CAT activity, we showed that the groEL promoter was up-regulated during infection of the J774 macrophage line. The immune response to SIV capsid was assessed in Balb/c mice given one oral dose of vaccine. A local mucosal secretory IgA response against SIV capsid was detected but no systemic antibody response to the same antigen. A systemic CTL response was detected as early as 28 days to as late as 70 days post-immunization. CTL activity was MHC restricted (H-2d) and was mediated by CD3+, CD8+, CD4- T-lymphocytes. These results indicate that with only one oral dose of recombinant Salmonella using the APC vector, a systemic CTL response and a mucosal secretory response against the SIV capsid antigen are elicited in a mouse model.

Animals↗
PubMed2006

Protection of chickens against avian hepatitis E virus (avian HEV) infection by immunization with recombinant avian HEV capsid protein.

Avian hepatitis E virus (avian HEV) is an emerging virus associated with hepatitis-splenomegaly syndrome in chickens in North America. Avian HEV is genetically and antigenically related to human HEV, the causative agent of hepatitis E in humans. In the lack of a practical animal model, avian HEV infection in chickens has been used as a model to study human HEV replication and pathogenesis. A 32 kDa recombinant ORF2 capsid protein of avian HEV expressed in Escherichia coli was found having similar antigenic structure as that of human HEV containing major neutralizing epitopes. To determine if the capsid protein of avian HEV can be used as a vaccine, 20 chickens were immunized with purified avian HEV recombinant protein with aluminum as adjuvant and another 20 chickens were mock immunized with KLH precipitated in aluminum as controls. Both groups of chickens were subsequently challenged with avian HEV. All the tested mock-immunized control chickens developed typical avian HEV infection characterized by viremia, fecal virus shedding and seroconversion to avian HEV antibodies. Gross hepatic lesions were also found in portion of these chickens. In contrast, none of the tested chickens immunized with avian HEV capsid protein had detectable viremia, fecal virus shedding or observable gross hepatitis lesions. The results from this study suggested that immunization of chickens with avian HEV recombinant ORF2 capsid protein with aluminum as adjuvant can induce protective immunity against avian HEV infection. Chickens are a useful small animal model to study anti-HEV immunity and pathogenesis.

Animals↗
PubMed2006

The effect of capsid mutations on HIV-1 uncoating.

Efficient uncoating requires not only an optimal cellular environment, but also some intrinsic properties of the viral capsid protein itself. Using an in vitro uncoating model, we demonstrated that substitution of each serine residue with alanine at the three major phosphorylation sites of HIV-1 capsid protein, i.e. Ser-109, Ser-149 and Ser-178, could significantly reduce uncoating activity of purified core particles. We also showed that the core stability of mutant viruses was lower than that of the wild-type virus so that the lack of efficient uncoating of each mutant could not be due to an increase in capsid physical stability. However, serine-to-aspartic acid mutation to mimic the negative charge of phosphor-serine could not restore either uncoating activity or infectivity, and treatment of purified core particles with a phosphatase did not alter the uncoating activity. Our data indicated that mutations at phosphoacceptor sites of capsid disturbed the uncoating mechanism, but the defect may not be directly caused by the lack of phosphate on the core particles undergoing uncoating.

Amino Acid Substitution↗
PubMed2019

A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism.

Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid structures on a large scale using only a single screening round in vivo. The approach stands in contrast to previous methods that require multiple generations of enrichment. With the BRAVE approach, each virus particle displays a peptide, derived from a protein, of known function on the AAV capsid surface, and a unique molecular barcode in the packaged genome. The sequencing of RNA-expressed barcodes from a single-generation in vivo screen allows the mapping of putative binding sequences from hundreds of proteins simultaneously. Using the BRAVE approach and hidden Markov model-based clustering, we present 25 synthetic capsid variants with refined properties, such as retrograde axonal transport in specific subtypes of neurons, as shown for both rodent and human dopaminergic neurons.

barcoding↗
PubMed1993

Detection of simian immunodeficiency virus and human immunodeficiency virus type 2 capsid antigens by a monoclonal antibody-based antigen capture assay.

We have tested the ability of a monoclonal antibody-based simian immunodeficiency virus (SIV) p27 capsid antigen assay to detect SIV antigen in supernatants from a variety of infected cell cultures. The antigen capture assay has a sensitivity of approximately 30 pg of SIV p27 capsid antigen/ml. The assay detected SIV p27 capsid antigen in cell culture supernatants from all six strains tested, detected the replication of SIV following the inoculation of the virus in peripheral blood mononuclear cell cultures earlier compared to reverse transcriptase assay, and was more sensitive in detection of the SIV antigen compared to human immunodeficiency virus type 1 (HIV-1) antigen capture assays. The SIV antigen capture assay was used to detect SIV antigen from serum samples and tissue cultures from eight of eight SIVB670-infected rhesus macaques (Macaca mulatta). Similar samples from four control rhesus macaques were negative when tested by the assay. The SIV antigen was detected in virus-infected monkeys during early time periods following inoculation (1 to 3 weeks) or during episodes of CD4+ lymphocytopenia and clinically evident disease. In addition, the SIV antigen capture assay positively identified each of three different HIV-2 strains in cell culture supernatants. The SIV antigen capture assay provides a sensitive and specific method to monitor SIV and HIV-2 capsid antigen in cell cultures and from infected animals. The assay will be an important tool in the utilization of SIV and HIV-2 primate models for HIV-induced acquired immunodeficiency disease syndrome.

Animals↗
PubMed1983

Monospecific antisera against capsid polypeptides of poliovirus type 1 distinguish antigenic structures of poliovirus proteins.

Pure poliovirus polypeptides, namely VP1, VP2, VP3 and VP4, have been isolated by isoelectric focusing after dissociation of poliovirus by urea. When injected into rabbits, all four polypeptides produced monospecific antisera which were used for the characterization of poliovirus particles and poliovirus-infected cells. The specificity of these antisera was determined by immunoprecipitation of polypeptides obtained by dissociation of poliovirus with SDS, followed by characterization by polyacrylamide gel electrophoresis. The antisera revealed differences in the antigenic sites of native poliovirus particles, heated poliovirus particles and naturally occurring empty capsids. Only VP3 antiserum reacted with native poliovirus and showed some neutralization, whereas all antisera precipitated heated virus and empty capsids. These antisera reacted also with the appropriate precursors of the capsid polypeptides demonstrating their usefulness for an analysis of the cleavage pathway by monospecific antibodies and revealed a second protomer (90 kilodalton) polypeptide for the capsid proteins of poliovirus particles.

Antibodies, Viral↗
PubMed1993

Determination and refinement of the canine parvovirus empty-capsid structure.

The canine parvovirus (CPV) empty-capsid structure has been determined and refined to 3.0 A resolution in the tetragonal space group P4(3)2(1)2 with cell dimensions a = b = 254.5 and c = 795.0 A. The successful structure determination shows that reasonably good diffraction data were obtained in spite of the very long c axis. The structure was solved by molecular replacement using the electron density of CPV full particles in a monoclinic space group. The phases were refined by non-crystallographic symmetry averaging. The structure refinement was carried out by using the programs PROLSQ and X-PLOR. The final R factor for the structure that included 85 water molecules per icosahedral asymmetric unit was 21.1% for reflections between 6.0 and 3.0 A resolution with an r.m.s. deviation of bond lengths of 0.020 A from ideal values. The structure of CPV empty capsids showed conformational differences with respect to full capsids at a region where icosahedrally ordered DNA in full particles interacts with the capsid protein. It also confirmed the absence of density along the fivefold axis in the CPV empty-particle structure in contrast to the situation in CPV full particles.

Journal Article↗
PubMed1993

Differentiation of Two Abortive Mechanisms by Using Monoclonal Antibodies Directed toward Lactococcal Bacteriophage Capsid Proteins.

Monoclonal antibodies were used to monitor the accumulation of the major capsid protein of the lactococcal small isometric bacteriophage u136 (P335 species) over the course of a one-step growth curve. A sandwich enzyme-linked immunosorbent assay was then used to distinguish two abortive phage resistance mechanisms, Hsp and Prf. Capsid protein production of u136 was almost totally inhibited by the Hsp-induced abortive mechanism, supporting previous data that this mechanism blocks phage DNA replication. Prf-induced abortive infection only partially (50%) inhibited capsid protein production, suggesting that this mechanism targets some other point, perhaps within transcription or translation processes. The results confirmed that Hsp and Prf act at different targets in the phage lytic cycle. Use of monoclonal antibodies also demonstrated that production of the major capsid protein is a nonlimiting step in the lytic cycle of lactococcal phage u136.

Journal Article↗
PubMed1973

Electrophoretic differences in the capsid proteins of simian virus 40 plaque mutants.

The structural proteins of three mutants of simian virus 40 (SV40) which differ in plaque size, temperature sensitivity, oncogenicity, host cell restriction, and immunological properties were studied. The polypeptide components of these SV40 strains could not be distinguished by their polyacrylamide gel electrophoretic patterns. When the dissociated virions of two of the mutants were analyzed by the isoelectric focusing technique in a urea gradient, the capsid protein peaks were found to differ significantly in their isoelectric points. The capsid protein of the small-plaque mutant had an isoelectric point of pH 6.51 as compared with pH 6.28 for the large-plaque strain. Isoelectric focusing of the isolated capsid protein revealed three components, a single major subunit and two minor forms. The coat proteins of two of the mutants, small-plaque and minute-plaque strains, were indistinguishable by this technique. The capsid protein peaks obtained by isoelectric focusing were further analyzed by polyacryalmide gel electrophoresis.

Animals↗
PubMed1968

Effect of formamide on the capsid morphology of adenovirus types 4 and 7.

Treatment of adenovirus types 4 and 7 with formamide disrupted the virions, degrading the capsids into predominantly single capsomers. As shown by electron microscopic observation, disruption proceeded in the following sequence: (i) reduction of the electron density of the virions, suggesting release of an internal component; (ii) progressive cleavage of the capsid into two or more segments and the formation (type 7 only) of capsomer "sheets"; (iii) final cleavage of the capsid into single or groups of a few capsomers. The sequence appeared similar for both adenoviruses; for both types, the rate and extent of disruption were dependent on the formamide concentration, but type 7 was more easily disrupted than type 4 by short-term (5 to 10 sec) treatment at the low (10%) concentration. At 30% formamide, the intercapsomer bonds of either type were fully cleaved, and the capsids were completely degraded into predominantly single capsomers. Pretreatment with formaldehyde did not prevent this degradation. Under suitable conditions, virus-derived remnants can be observed among the breakdown products. These remnants have been shorn of capsomers and presumably represent intact internal nucleoprotein.

Adenoviridae↗
PubMed1971

Synthesis of capsid and noncapsid viral proteins in response to encephalomyocarditis virus ribonucleic acid in animal cell-free systems.

The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of (35)S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides.

Animals↗
PubMed1997

Humoral immune response to the capsid components of recombinant adenoviruses: routes of immunization modulate virus-induced Ig subclass shifts.

This study examines in detail the capsid-specific humoral immune response of BALB/c mice after one single injection of a replication-defective adenovirus. Two routes of immunization, intravenous (i.v.) and intraperitoneal (i.p.), were compared for the response induced against the adenovirus particle and the three major components of the viral capsid, hexon, penton base, and fiber. A single immunization with the replication-defective adenovirus induces a long and persistent humoral response specific for the virus. However, the molecular components of the viral capsid are differentially recognized depending on the route of immunization. The sera from mice immunized i.p. recognized only the hexon protein and a preferential switch to the IgG2a subclass was obtained which remained stable 100 days post-immunization. The sera obtained from mice immunized i.v. gave a more complex response. At the beginning of the response, an isotype bias toward the IgG2a subclass was observed, but the isotype distribution changed during the whole period of the response. Neutralizing activity was maximum 45 days after immunization by both routes, and no activity was detectable after 3 months. However, the i.v. serum displayed a higher neutralizing activity than the i.p. serum. The IgM antiviral antibodies appeared to be an important component of the neutralizing activity, and the two routes of immunization do not induce the same IgG isotypes to neutralize viral infectivity. Extension of these findings to human gene therapy using recombinant adenoviruses may help to characterize the precise viral protein targets of neutralizing antibodies.

Adenoviruses, Human↗
PubMed1991

Host range determinants located on the interior of the poliovirus capsid.

The inability of certain poliovirus strains to infect mice can be overcome by the expression of human poliovirus receptors in mice or by the presence of a particular amino acid sequence of the B-C loop of the viral capsid protein VP1. We have identified changes in an additional capsid structure that permit host-restricted poliovirus strains to infect mice. Variants of the mouse-virulent P2/Lansing strain were constructed containing amino acid changes, deletions and insertions in the B-C loop of VP1. These variants were attenuated in mice, demonstrating the importance of the B-C loop sequence in host range. Passage of two of the B-C loop variants in mice led to the selection of viruses that were substantially more virulent. The increased neurovirulence of these strains was mapped to two different suppressor mutations in the N-terminus of VP1. Whereas the B-C loop of VP1 is highly exposed on the surface of the capsid, near the five-fold axis of symmetry, the suppressor mutations are in the interior of the virion, near the three-fold axis. Introduction of the suppressor mutations into the genome of the mouse-avirulent P1/Mahoney strain resulted in neurovirulent viruses, demonstrating that the P2/Lansing B-C loop sequence is not required to infect mice. Because the internal host range determinants are in a structure known to be important in conformational transitions of the virion, the host range of poliovirus may be determined by the ability of virions to undergo transitions catalyzed by cell receptors.

Alleles↗
PubMed2005

Quaternary structure is critical for protein display on capsid-like particles (CLPs): efficient generation of hepatitis B virus CLPs presenting monomeric but not dimeric and tetrameric fluorescent proteins.

Self-organizing assemblies such as viral capsids may be used as symmetrical molecular platforms for the display of heterologous sequences, with applications ranging from vaccines to structural studies. The 183-amino-acid hepatitis B virus (HBV) core protein assembles spontaneously into icosahedral capsid-like particles (CLPs). The most exposed, and most immunogenic, substructure on the CLPs is a small loop that connects two long antiparallel alpha-helices which act as dimerization interface. Ninety (90) or 120 dimers multimerize into the capsid; the four-helix bundles formed by the dimers protrude as spikes from the surface. We recently demonstrated that the entire enhanced green fluorescent protein (eGFP) can be inserted into this loop, yielding CLPs that natively displayed eGFP on their surface. The central location of the insertion site requires that any insert be fixed to the carrier via both termini, with corresponding restrictions regarding insert size and structure. eGFP obviously satisfied these criteria but, surprisingly, all attempts to produce CLPs with the isostructural red fluorescent proteins DsRed1, DsRed2, and HcRed failed. Suspecting their oligomerization tendency to be responsible, we generated fusions containing instead monomeric yellow, cyan, and red fluorescent proteins (mYFP, mCFP and mRFP1). This strongly increased the yields of YFP and CFP-CLPs, and it allowed for the first time to efficiently generate red fluorescent CLPs. Hence insert quaternary structure is a highly critical factor for CLP assembly. These data have important implications for the rational design of self-assembling fusion proteins.

Bacterial Proteins↗
PubMed1997

Projection structure of VP6, the rotavirus inner capsid protein, and comparison with bluetongue VP7.

The rotavirus nucleocapsid protein (VP6) is the major structural protein of inner capsid particles (ICP). VP6 is essential for RNA transcription and binds to a virally encoded glycoprotein receptor (NSP4) involved in the rotavirus assembly pathway. To explore the structure of VP6, two-dimensional (2D) crystals of VP6 were generated and examined by electron microscopy and image processing. Fourier transforms computed from low-dose images of negatively stained 2D VP6 crystals displayed complete data to 13 A resolution for p6 plane group symmetry. To correct for the resolution dependent fall-off of the amplitudes derived from electron microscopic images, the rotavirus VP6 amplitudes were scaled to the bluetongue VP7 amplitudes derived from the atomic model by applying a B factor of -360 A-2. The unit cell (a=b=101(+/-2)A, gamma=120(+/-1) degrees) contains two VP6 trimers, each composed of three roughly circular subunits approximately 30 A in diameter. The trimeric organization of VP6 is similar to the oligomeric structure of VP6 when assembled in T=13l icosahedral inner capsid particles at 25 to 40 A resolution. However, a channel at the center of the trimer is better resolved in our map at 15 A resolution. The projection structure of rotavirus VP6 was compared to the homologous protein (VP7) of bluetongue virus, which is also a member of the family of Reoviridae. Notably, both VP6 and bluetongue VP7 assemble as 260 capsomers on the surface of the inner capsid. To compare VP6 and VP7, a projection map of bluetongue VP7 at 15 A resolution was generated using the atomic model derived by X-ray crystallography. VP6 and VP7 both exhibit a trimeric organization with a central channel, even though the alignment identity between the 45 kDa VP6 and the 38 kDa VP7 primary sequences is only 12%. The ability of VP6 to form well-ordered 2D crystals should enable a higher resolution structure analysis by cryo-electron microscopy that will extend our understanding of the icosahedral ICP structure, clarify the mechanism by which VP6 interacts with the NSP4 receptor, and allow a more detailed comparison of VP6 and VP7.

Antigens, Viral↗
PubMed1998

The SV40 capsid protein VP3 cooperates with the cellular transcription factor Sp1 in DNA-binding and in regulating viral promoter activity.

Chromatin structure and protein-protein interactions play an important role in eukaryotic gene function. Nucleosomal rearrangement at the simian virus 40 (SV40) regulatory region occurs at the late stages of the viral life cycle preceding viral assembly. The SV40 capsid proteins are required for this nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found significant cooperativity between Sp1 and VP3 in specific DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These findings have led us to conclude that the novel host-viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.

Base Sequence↗
PubMed2001

Structural studies on the empty capsids of Physalis mottle virus.

The three-dimensional crystal structure of the empty capsid of Physalis mottle tymovirus has been determined to 3.2 A resolution. The empty capsids crystallized in the space group P1, leading to 60-fold non-crystallographic redundancy. The known structure of Physalis mottle virus was used as a phasing model to initiate the structure determination by real-space electron-density averaging. The main differences between the structures of the native and the empty capsids were in residues 10 to 28 of the A-subunit, residues 1 to 9 of the B-subunit and residues 1 to 5 of the C-subunit, which are ordered only in the native virus particles. An analysis of the subunit disposition reveals that the virus has expanded radially outward by approximately 1.8 A in the empty particles. The A-subunits move in a direction that makes 10 degrees to the icosahedral 5-fold axes of symmetry. The B and C-subunits move along vectors making 12 degrees and 15 degrees to the quasi 6-fold axes. The quaternary organization of the pentameric and hexameric capsomeres are not altered significantly. However, the pentamer-hexamer contacts are reduced. Therefore, encapsidation of RNA appears to cause a reduction in the particle radius concomittant with the ordering of the N-terminal arm in the three subunits. These structural changes in Physalis mottle virus appear to be larger than the corresponding changes observed in viruses for which both the empty and full particle structures have been determined.

Binding Sites↗