Conditions of stability for the quaternary structure and antigenic activity of adenovirus hexon

The procedure of SDS-PAGE was modified by lowering the temperature of protein sample dissociation to allow the separation of denaturated adenoviral hexon chains and native hexon capsomers (trimers) in the same gel. By combining the modified SDS-PAGE with dot and blot radioimmunoassays, the range of stability of the simian adenovirus SA7 hexon quaternary structure and its antigenicity was studied against a number of physical and chemical agents known to dissociate and denaturate proteins. A perfect correlation was found between the hexon native quaternary structure (trimer) and its immunoreactivity with anti-hexon immunoglobulins. The pattern of hexon trimer stability to a wide spectrum of denaturants suggests that its subunits are held together, mainly by hydrophobic interactions, in such a way that the innersubunit contact regions make up the "hydrophobic core" of the hexon molecule.     

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.     

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.     

Studies on the molecular weight of the adenovirus type 2 hexon and its subunit

The molecular weight of the adenovirus type 2 hexon was calculated from sedimentation equilibrium, light scattering and sedimentation and diffusion experiments. The extinction coefficient, E_{1 cm}^1%, was determined to be 14.3 at 279 nm, from quantitative nitrogen and carbon analyses combined with the N,C content calculated from the amino acid composition. Other parameters determined were: the partial specific volume, $\text{}\text{?}\text{gn = 0.738}\text{cm}{}^3\text{g}{}^{\mathrm{?1}}$; the refractive index increment, $\text{(}\text{?n}\text{?c}\text{)}{}_{\mathrm{<mtext>T,P</mtext><mtext>,\mu </mtext>}}\text{= 0.193}\text{cm}{}^3\text{g}{}^{\mathrm{?1}}$ at 435.8 nm; the sedimentation coefficient, s_20,w⁰ = 13.0 S; and the diffusion constant, D_{20, w}⁰ = 3.32 × 10^?7 cm² s^?1. All molecular weights were between 355,000 and 363,000. Crystal density measurements were made on native and glutaraldehyde cross-linked crystals and the molecular weights calculated from these data were compared with the precise molecular weight determined by physico-chemical methods.Only one polypeptide of molecular weight 120,000 was found in reduced, or reduced and alkylated, hexon. Four or six organomercurial molecules were bound per 120,000 molecular weight of native hexon upon titration with 2-chloromercuri-4-nitrophenol and 2-chloromercuri-4,6-dinitrophenol, respectively. With 5,5′-dithiobis (2-nitrobenzoic acid) only one SH-group per 120,000 could be titrated in native hexon, but after denaturation in 1% sodium dodeeyl sulphate five more SH-groups reacted per 120,000 molecular weight. Thus there are three identical polypeptides of molecular weight 120,000 per hexon of total molecular weight 360,000.     

Purification and properties of the hexon antigen of canine adenovirus serotype CAV-1

The main antigen and immunogen of canine adenovirus type 1 (CAV-1) has been purified to near homogeneity from cultural fluid of a CAV-1-infected primary cell culture by hydrophobic and anion-exchange chromatography. The hexon native form (trimer) was shown to be resistant against denaturation by SDS under conditions of SDS-PAGE performed without heating the samples. The monomer chain of the CAV-1 hexon was apparently identical in terms of electrophoretic mobility with that of the previously sequenced BAV-3 hexon polypeptide (103 kDA). In blot enzyme immunoassay only native trimers of CAV-1 hexon were detected by cross-specific polyclonal and monoclonal anti-hexon antibodies.     

Primary structure of the murine adenovirus type 1 proteinase.

The DNA sequence of an open reading frame (ORF) corresponding to the murine adenovirus type 1 (Mav1) proteinase gene was determined. 1162 base pairs were sequenced from the downstream end of the SmaI-D Mav1 genomic fragment. The sequence defines the 204 amino acid proteinase, which apparently does not possess the usual L3 polyadenylation signal, but instead the sequence AAATAA. This gene is followed by a 147 amino acid C-terminal portion of the DNA-binding protein, encoded by the complementary strand.     

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.     

Characterization of a temperature-sensitive, hexon transport mutant of type 5 adenovirus.

Infection of KB cells at 39.5 degrees C with H5ts147, a temperature-sensitive (ts) mutant of type 5 adenovirus, resulted in the cytoplasmic accumulation of hexon antigen; all other virion proteins measured, however, were normally transported into the nucleus. Immunofluorescence techniques were used to study the intracellular location of viral proteins. Genetic studies revealed that H5ts147 was the single member of a nonoverlapping complementation group and occupied a unique locus on the adenovirus genetic map, distinct from mutants that failed to produce immunologically reactive hexons at 39.5 degrees C ("hexon-minus" mutants). Sedimentation studies of extracts of H5ts147-infected cells cultured and labeled at 39.5 degrees C revealed the production of 12S hexon capsomers (the native, trimeric structures), which were immunoprecipitable to the same extent as hexons synthesized in wild type (WT)-infected cells. In contrast, only 3.4S polypeptide chains were found in extracts of cells infected with the class of mutants unable to produce immunologically reactive hexon protein at 39.5 degrees C. Hexons synthesized in H5ts147-infected cells at 39.5 degrees C were capable of being assembled into virions, to the same extent as hexons synthesized in WT-infected cells, when the temperature was shifted down to the permissive temperature, 32 degrees C. Infectious virus production was initiated within 2 to 6 h after shift-down to 32 degrees C; de novo protein synthesis was required to allow this increase in viral titer. If ts147-infected cells were shifted up to 39.5 degrees C late in the viral multiplication cycle, viral production was arrested within 1 to 2 h. The kinetics of shutoff was similar to that of a WT-infected culture treated with cycloheximide at the time of shift-up. The P-VI nonvirion polypeptide, the precursor to virion protein VI, was unstable at 39.5 degrees C, whereas the hexon polypeptide was not degraded during the chase. It appears that there is a structural requirement for the transport of hexons into the nucleus more stringent than the acquisition of immunological reactivity and folding into the 12S form.     

Distribution of H type 1 and H type 2 antigenic determinants in human sera and saliva.

A radioimmunoassay specific for the H type 1 antigenic determinant demonstrated that the H type 1 antigen is under the strict control of the Se gene in both serum and saliva. Similar amounts of H type 1 antigenic determinants were found in saliva from Se/-, le/le donors and in saliva from Se/-, Le/- donors. However, sera from Se/-, le/le donors were about 100 times more efficient in inhibiting the H type 1 assay than were sera from Se/-, Le/- donors. A radioimmunoassay, based on the binding of Ulex europaeus with the H type 2 antigenic determinant, showed that all the H type 2 antigen in saliva is under the control of the Se gene, while only one-third of the H type 2 antigen present in serum is under the control of this gene. The remaining two-thirds of H type 2 antigen in sera is independent of the ABH secretor status of the donor. The amount of H type 2 antigen in both serum and saliva is independent of the Le gene. These results are compatible with the existence of two alpha (1 leads to 2) fucosyl-transferases but suggest that the enzyme of epithelial origin, coded by the Se gene, should be able to transform both type 1 and type 2 natural substrates, while the enzyme of mesodermic origin, coded by the H gene, would work preferentially on the natural type 2 substrates.