Stability of the structure and antigenic determinants of adenovirus type 1 native hexon to proteases

Hexon capsomers of human adenovirus type 1 (h1) labeled by iodine 125 were digested in a native state (trimers) by trypsin, chymotrypsin or papain, and the resulting hydrolysates were analyzed by SDS-PAGE. In each case, a discrete and temporally stable pattern of relatively large fragments was revealed. The degree of hexon polypeptide hydrolysis was maximal for papain, intermediate for chymotrypsin and minimal for trypsin, the largest fragments in the digest being 32, 40 and 80 kD, respectively. At room temperature, all the electrophoretically discernible hexon proteolytical fragments were held together in structures resembling intact hexon trimers and could be regarded as "hexon cores", of which papain hexon cores were the most stable during SDS-PAGE. Radioimmunoprecipitation analysis revealed a complete absence of native hexon antigenicity in thermodenaturated fragments of hexon protease digests, while native trypsin, chymotrypsin and papain hexon cores could be precipitated by hexon-specific antibodies. The immunoprecipitated material contained all of the hexon fragments found in appropriate hexon cores and retained the structure of the original cores. Trypsin, chymotrypsin and papain hexon cores were shown to possess at least part of native Ad h1 hexon antigenic determinants of each of the following specificities: species-specific (epsilon), cross-reactive with hexon of human adenoviruses (h3 and h6), simian adenovirus (sim 16), bovine adenoviruses (bos 3 and bos 7) and avian adenovirus (Aviadenovirus gal 1 or CELO). Thus, the full spectrum of known hexon antigenic determinants (species-specific to intergenus-crossreactive) is at least portly stable against protease attack of native hexon capsomers.     

The possibilities for enhancing the immunogenicity of adenovirus hexon

Synthetic polyelectrolytes and muramyldipeptide derivatives are tested as immunostimulators. It is established that the level of antibody-forming cells and specific (virus-neutralizing and precipitating) antibodies considerably rises during the immunization of human adenovirus I hexon in the combination with N-acetylglucosaminyl-muramyldipeptide and conjugation with a copolymer including acrylic acid and N-vinylpyrrolidone. A more expressed intensification of the antibody-formation is observed with the use of polyelectrolytes.     

The N-terminal peptide of the hexon of human adenovirus type 2: its chemical synthesis and antigenic properties]

Peptides corresponding to the N-terminal section of protein of a hexone of the 2nd type human adenovirus have been synthesized. Being conjugated with a carrier of BSA they induced formation of antibodies which reacted both with peptides and with viral proteins. Sera to native proteins were also bound to synthetic peptides. Immunogenicity strengthening in peptides conjugated with synthetic polyelectrolytes is shown. The N-terminal section of hexon is supposed to participate in formation of an antigenic determinant possessing group specificity.     

Consideration of the three dimensional structure of the adenovirus hexon from electron microscopy and computer modelling

A three-dimensional model of the adenovirus hexon has been built up from electron microscopic observations and information obtained by computer modelling. It has been shown that the hexons (in upright position) are negatively stained from below and that stain layer after drying is only 1–2nm high. Thus the top, i.e. the external part, of the hexon is triangular, the waist is hexagonal and the bottom is ‘round’ with a large axial hole. The top is twisted through 30° with respect to the waist. This morphological polarity is accompanied by differences in physico-chemical properties: the top appears to be negatively charged at neutral pH, whereas the bottom is rather hydrophobic. Because of the hexagonal region at the waist of the hexon the virus capsid becomes sealed allowing passage of small molecules only, presumably via a narrow central channel.     

Identification of sites in adenovirus hexon for foreign peptide incorporation

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.     

Capsid-like arrays in crystals of chimpanzee adenovirus hexon

The major coat protein, hexon, from a chimpanzee adenovirus (AdC68) is of interest as a target for vaccine vector modification. AdC68 hexon has been crystallized in the orthorhombic space group C222 with unit cell dimensions of a = 90.8 A, b = 433.0 A, c = 159.3 A, and one trimer (3 x 104,942 Da) in the asymmetric unit. The crystals diffract to 2.1 A resolution. Initial studies reveal that the molecular arrangement is quite unlike that in hexon crystals for human adenovirus. In the AdC68 crystals, hexon trimers are parallel and pack closely in two-dimensional continuous arrays similar to those formed on electron microscope grids. The AdC68 crystals are the first in which adenovirus hexon has molecular interactions that mimic those used in constructing the viral capsid.     

The structure of the adenovirus capsid. II. The packing symmetry of hexon and its implications for viral architecture

The orientation and location of the 240 hexons comprising the outer protein shell of adenovirus have been determined. Electron micrographs of the capsid and its fragments were inspected for the features of hexon known from the X-ray crystallographic model as described in the accompanying paper. A capsid model is proposed with each facet comprising a small p3 net of 12 hexons, arranged as a triangular sextet with three outer hexon pairs. The sextet is centrally placed about the icosahedral threefold axis, with its edges parallel to those of the facet. The outer pairs project over the facet edges on one side of the icosahedral twofold axes at each edge. The model capsid is defined by the underlying icosahedron, of edge 445 A, upon which hexons are arranged. The hexons are thus bounded by icosahedra with insphere radii of 336 A and 452 A. A quartet of hexons forms the asymmetric unit of an icosahedral hexon shell, which can be closed by the addition of pentons at the 12 vertices. Considering the hexon trimer as a complex structure unit, its interactions in the four topologically distinct environments are very similar, with conservation of at least two-thirds of the inter-hexon bonding. The crystal-like construction explains the flat facets and sharp edges characteristic of adenovirus. Larger "adenovirus-like" capsids of any size could be formed using only one additional topologically different environment. The construction of adenovirus illustrates how an impenetrable protein shell can be formed, with highly conserved intermolecular bonding, by using the geometry of an oligomeric structure unit and symmetry additional to that of the icosahedral point group. This contrasts with the manner suggested by Caspar & Klug (1962), in which the polypeptide is the structure unit, and for which the number of possible bonding configurations required of a structure unit tends to infinity as the continuously curved capsid increases in size. The known structures of polyoma and the plant viruses with triangulation number equal to 3 are evaluated in terms of hexamer-pentamer packing, and evidence is presented for the existence of larger subunits than the polypeptide in both cases. It is suggested that spontaneous assembly can occur only when exact icosahedral symmetry relates structure units or sub-assemblies, which would themselves have been formed by self-limiting closed interactions. Without such symmetry, the presence of scaffolding proteins or nucleic acid is necessary to limit aggregation.     

Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues.

The first full-length hexon protein DNA and deduced amino acid sequences of a subgenus D adenovirus (AV) were determined from candidate AV48 (85-0844). Comprehensive comparison of this sequence with hexon protein sequences from human subgenera A, B, C, D, F, bovine AV3, and mouse AV1 revealed seven discrete hypervariable regions (HVRs) among the 250 variable residues in loops 1 and 2. These regions differed in length between serotypes, from 2 to 38 residues, and contained > 00% of hexon serotype-specific residues among human serotypes. Alignment with the published crystal structure of AV2 established the location and structure of the type-specific regions. Five HVRs were shown to be part of linear loops on the exposed surfaces of the protein, analogous to the serotype-specific loops or "puffs" in picornavirus capsid proteins. The HVRs were supported by a common framework of conserved residues, of which 68 to 75% were hydrophobic. Unique sequences were limited to the seven HVRs, so that one or more of these regions contain the type-specific neutralization epitopes. A neutralizing AV48 hexon-specific antiserum recognized linear peptides that corresponded to six HVRs by enzyme immunoassay. Affinity-purification removal of all peptide-reactive antibodies did not significantly decrease the neutralization titer. Eluted peptide-reactive antibodies did not neutralize. Human antisera that neutralized AV48 did not recognize linear peptides. Purified trimeric native hexon inhibited neutralization, but monomeric heat-denatured hexon did not. We conclude that the AV48 neutralization epitope(s) is complex and conformational.     

An acetylated N-terminus of adenovirus type 2 hexon protein

An acetylated N-terminus of adenovirus type 2 hexon protein was characterized using radioactively labeled protein and mass spectrometry. The labeled protein, obtained by synthesis in a medium containing ¹⁴C-acetate, was digested with proteolytic enzymes. A radioactive peptide, Acetyl-Ala-Thr-Pro-Ser, recovered in good yield, was the only N-terminal structure detected in the protein.     

Chimeric hexon HVRs protein reflects partial function of adenovirus

Adenovirus is widely used in gene therapy and vaccination as a viral vector, and its hypervariable regions (HVRs) on hexon are the main antigen recognition sites of adenovirus. The modification of this area by genetic engineering will change the antigenic specificity of the virus. In addition, recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus serotype 5-mediated liver transduction in vivo. The binding site of adenovirus to FX is the HVRs on hexon. By constructing five proteins containing chimeric HVRs from different adenovirus serotypes, we focused on the antigenic specificity and the affinity for FX of these proteins compared with the corresponding viruses. Our data showed that HVR5 and HVR7 had only a part of hexon activity to neutralizing antibodies (NAbs) compared with the complete activity of HVR1-7. Results also demonstrated a differential high-affinity interaction of the HVRs proteins with FX and indicated that HVRs protein had a similar binding ability with corresponding adenovirus serotype. These results highlighted some properties of chimeric HVRs proteins and revealed the influence on the structure and function of hexon proteins and adenovirus resulting from the HVRs.     

The location of the genes coding for hexon and fiber proteins in adenovirus DNA.

A serological analysis has been made of the capsid antigens hexon and fiber from 17 Ad5-Ad2+ND1 recombinants that enables us to determine the phenotype of the recombinants. By correlation of this data with the genetic and physical maps of the adenovirus genome, obtained by recombination and restriction endonuclease analysis, the genes coding for the hexon and fiber have been assigned to specific locations on the adenovirus DNA.     

The relationship between quaternary structure and enzyme activity

Methods have been developed to study the influence of quaternary structure on enzyme activity. Some enzymes which normally exist as stable oligomers remain catalytically active when the subunits are dissociated by artificial means.