Biomedical subjects
Publications and source records attributed to E Adám.
The mutual rotational orientation of the three hexon polypeptide subunits of the peripentonal hexons has been determined in relation to each other, to the penton and to the neighbouring hexons. The possible mutual orientation of all the polypeptide subunits of the adenovirus-building hexon capsomers are presented on a tentative virus model.
Three-thousand hybridoma clones were produced using crystallized hexon of human adenovirus type 1 for immunization. ELISA revealed 69 homospecific antibody producing clones. The monoclonals were reactive with both crystallized and non-crystallized purified soluble hexon preparations, as well as with the hexons derived from purified virions. Hexon preparations of 10 different human adenovirus species (members of 4 different subgenera) were used for the characterization of 33 monoclonal antibodies. ELISA and passive haemagglutination (HA) tests revealed 6 and 15 distinct reactivity patterns (RP) i.e. intertype cross-reaction of a given antibody with the panel of test-antigens, respectively. Four of these RP-s were identical by both techniques. Significant quantitative differences existed between extinction values in ELISA and HA-titres. The results indicate the presence of identical epitopes as well as epitypic relationships among the hexons. The common epitopes and the epitypic relationship seem to be characterized by 16 different interspecies specificities under the experimental conditions, which are independent of subgenus boundaries and are demonstrable in different combinations on the hexons of adenovirus species belonging either to the same or to different subgenera.
The interhexonal space within the adenovirus capsid is bridged by interhexonal connective elements. These linking components have been detected also between pentons and the five peripentonal hexons surrounding them. According to the results obtained by Markham's rotational integration technique the connective elements between the capsomers are interlacing the whole virion. It could be confirmed in several cases that the linking elements consisted of two approximately parallel elements. This means that each hexon is linked by six times two parallel linking components with its six nearest neighbours in the virus capsid.
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With direct analysis of electron micrographs and with Markham's rotational integration technique we have demonstrated that in the adenovirus capsid the triangular profile of the hexon-end facing are formed by one polypeptide each and the sides by two neighbouring polypeptides. The mutual rotational and spatial orientation of the three hexon polypeptide subunits has been determined in relation to each other and to the pentons. Two polypeptides of the peripentonal hexons are oriented towards the penton. A "one-to-one" pattern was found in relation to two peripentonal hexons, "one-to-two" to two of the three neighbouring hexons and "two-to-two" to one hexon. "One-to-two" orientation is the general rule in the GONs. The mutual orientation between the hexons of the neighbouring GONs being in connection with each other and that of the polypeptide subunits of the peripentonal hexons is characterized by the regular alternation of the "one-to-two", "one-to-one" and "two-to-two" orientation. The mutual orientation between the hexons at different points of the capsid has been studied in model experiments and was characterized by the angle enclosed by the longitudinal axis of the given hexon and that of the other hexon in open position towards the virion surface, if the longitudinal axis of the hexons of the triangular faces are perpendicular to the plane of the face and those of the edge-hexons perpendicular to the plane of the edge and the longitudinal axis of the penton faces in radial direction the centre of the virion. In this way the longitudinal axes of the peripentonal hexons enclose with that of the penton an angle of approximately 32 degrees and with each other approximately 36 degrees. The longitudinal axis of the edge-forming hexons shows a deviation of approximately 21 degrees in relation to the longitudinal axes of hexons situated on the neighbouring triangular faces. We present on a tentative virus model the mutual rotational and spatial orientation of all the polypeptide subunits of the adenovirus-building hexon capsomers. This corresponds to the two-, three- and fivefold rotational symmetry characteristic of the icosahedral capsid.
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Two distinct profiles of hexon end parts were detected in two-dimensional adenovirus hexon crystalline arrays: (i) hexons displaying three approximately oblong subunits around a triangular hole, and (ii) triangular hexons showing three main subunits with Y-shaped slits in their centers. The rest of the hexons seemed to be approximately ring-shaped with a roundish hole at their centers. Direct examination of electron micrographs and their optical diffraction patterns as well as Markham's rotational integration technique permitted the establishment of the mutual orientation of hexons and of polypeptide subunits within small, distinct parts of the crystalline array. The position of the hexons is such that one of the polypeptide subunits of a given hexon is situated nearest to the two other polypeptides of the neighboring hexon ('one-to-two' linkage system).
Fine interhexonal connections were observed in high resolution electron micrographs of tight two-dimensional hexagonal crystalline arrays. Intrahexonal connections were found among the three polypeptides constituting the hexons. The length of the interhexonal connections was 2.73 mm, and their diameter 1.2 nm; that of the intrahexonal connections were 1.05 nm and 0.75 nm, respectively. The orientation of the hexons in a two-dimensional crystalline array corresponded to a corner to edge conjunction. One side of each of the three polypeptides, constituting a triangular hexon, corresponded to the sides of that hexon; their points of contact were the corners. A tentative diagram of the molecular arrangement of hexon polypeptides and of interhexonal connections was drawn up following analysis of the electron micrographs. It showed that parallel connections run to both ends of each polypeptide of a hexon, connected to one of the end parts of the two nearest polypeptides of the next hexon, as follows from the threefold symmetry. Thus, every hexon is joined to its six neighbours by six times two parallel connections. Two pairs of parallel connections enclose an angle of approximately 60 degrees.
Three profiles of hexons were detected om two-dimensional adenovirus crystalline arrays: (i) approximately ringwise closed hexons with a roundish hole in their centres and with 2-4 electron dense spots in the wall of the rings; (ii) hexons, consisting of three approximately oblong polypeptide enclosing a triangular hole; (iii) triangular hexons, containing three main polypeptides with an Y-shaped slit, instead of a hole, in their centres. Following the examination of directly or computer corrected electron micrographs a tentative model has been developed on the possible mutual "rotation" orientation of hexon polypeptides within the two-dimensional crystalline array. The position of hexons is such that the longer side of a polypeptide of each hexon is next to the end of two polypeptides of its neighbouring hexon, i.e. one polypeptide is linked to two other ones. An irregularity evolves in this "one-to-two" linkage system by the rotation of a hexon, the maximum rotation being 60% degrees, as follows from the hexon's threefold symmetry. The presence of lying hexons and of hexons of different contours, points to irregularities in "vertical" orientation, with the turn reaching even 90 degrees or 180 degrees, i.e. the hexons might be connected in such a way that their originally external or internal parts are facing identical directions.
Highly purified soluble adenovirus type 1 hexons were crystallized by dialyzing against 0.5 M acetate buffer, pH 4.5, for 3 days. Crystals of tetrahedral shape were formed after storage at 4 degrees within 1--10 days and ranged in size from 0.01 to 0.1 mm. In early phase of crystallization, diffusely dispersed hexons were observed by electron microscopy. Later, continuous monolayers of two-dimensional hexon paracrystalline arrays developed. Direct analysis of the electron micrographs and their optical diffraction patterns showed that the structure of the dense two-dimensional crystalline arrays of hexon capsomers was characterized by slightly skew and irregular hexagonal packing. Multilayered microcrystals with regular linear internal structure were also found. In addition, unique forms of aggregated hexons, such as holey hexon lattice and filamentous forms, were also found.
Soluble hexon of type 1 adenovirus was highly purified with different techniques and dialysed against 0.5 M acetate buffer. With this procedure tetrahedral crystals were produced from the soluble hexon capsomers of the virus capsid. Electron microscopic observation of the crystallization process, revealed the development of dense two-dimensional "crystal sheets" following dialysis, in homogeneous hexon preparations containing single hexons. No such formations were observed so far with other types. The occurrence of two-dimensional crystals decreased proportionally to the appearance of three-dimensional crystals, which refers to their possible role in the mechanism of three-dimensional crystal formation.
Separated, highly purified and concentrated adenovirus type 1 soluble hexon capsomers were crystallized by dialysis against 0.5 M acetate buffer. The crystallization process was followed electron microscopically. In the early phase of the crystallization, groups of a few hexons began to appear, then the two-dimensional crystal lattices grew gradually to a size of 1-2 micron. Simultaneously three-dimensional crystals of tetrahedral and prismatic shapes developed. The hexons in the two-dimensional crystal lattice formed regulator dense arrays corresponding to the hexagonal packing. Analysis of the crystal structure revealed 15-20% local irregularity (short range disorder) and about 10% deviation in the values of the lattice constant if determined from three different directions. The average lattice constant values showed considerable differences in different preparations. Angles formed by non-parallel hexon rows deviated by a few degrees from the regular hexagonal order. Consequently, the position of the hexons in dense two-dimensional crystals was found slightly skew and irregular, although each unit stayed within a certain distance as compared to its equilibrium position defined theoretically in the network. Dislocations were frequently found to disturb the regular arrays. The extra hexon row developing between two rows deverted them from their original direction. At these sites the crystal lattice slanted and the dense array of the hexons loosened. High resolution electron microscopy revealed fine linking structures between the hexons. In several cases the aggregated hexons failed to show a ring-like appearance, they were situated in lying--profile--position and the hexon-building polypeptide fibres became visible. The diameters of the hexons and the distance between them were measured in three directions and the size of the hexon-building polypeptides was determined as well.
The soluble proteins of adenovirus type 1 have been separated and purified. Their antigenic characteristics were compared in different precipitation experiments performed in electric field. Both two-dimension immune electrophoresis and rocket electrophoresis can successfully be applied for quick diagnostic purposes. Quantitative determination of virus proteins is also feasible by rocket electrophoresis. The isoelectric point values of hexon, penton and fibre were pI 4.55, pI 4.69 and pI 7.07, respectively. The amino acid composition of type 1 adenovirus and its capsid components was determined from separated and purified protein preparations. The former differed in amino acid composition from the tissues used for virus propagation.
Evidence is presented that the excess pool of penton complexes purified from adenovirus type 1-infected HEp-2 cells also carry an endonucleolytic activity. The effect was measured with both E. coli and adenovirus type 1 double-stranded DNA substrates. The use of agarose gels for assaying the endonuclease activity allowed the approximative calculation of the number of penton molecules which might produce one DNA double-strand break under the experimental conditions selected.
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Hexon antigens were separated from the cell-free supernatant of type 1 and type 8 adenovirus-infected cells by ammonium sulfate precipitation and purified by anion exchange chromatography, immune absorption, and exclusion chromatography. The antigens were compared with purified soluble hexons extracted from cells infected with the same adenovirus types. The procedure was found to be suitable for the preparation of pure, homogeneous type 1 hexons from the supernatant, but not for that of adenovirus type 8 hexons. Comparative studies showed no physico-chemical, immunological or electron microscopic differences between soluble hexons of extracellular and intracellular origin.