Adenovirus Assembly

Abstract

Cross-linking of adenovirus hexon capsomers by glutaraldehyde in vitro has been studied as a chemical probe for quaternary structure of hexon protein and virus capsid assembly. Glutaraldehyde cross-linking of hexons gave rise to three main oligomeric species, trimer, hexamer and nonamer, as analyzed on dodecylsulfate-acrylamide gels, which corresponded in terms of complete hexon capsomers, to single hexon capsomer, capsomer dimer and trimer. The major species was found to be the trimer of the hexon polypeptide unit, viz. the intramolecularly cross-linked hexon capsomer. This result indicated that intraoligomeric cross-linking took place more readily than the interoligomeric reactions. As shown by electron microscopy the spatial arrangement of the crosslinked hexon capsomer trimer was reminiscent of the structure of groups of three hexons clustered with %fold symmetry to form the groups of nine hexons isolated from disrupted adenovirus capsid.Virus assembly constitutes a crucial problem in molecular virology and many in vitro systems have been developed in an attempt to reproduce the morphogenesis of the virion within the host cell [l]. Very little is known about the mechanism of adenovirus assembly [2,3]. Hexon capsomer is the major subunit of the adenovirus icosahedral capsid and presents the advantage of being extractable from the infected cell as soluble native antigen, as well as the other capsid components, penton and fibre [4,5]. Although it has been reported that free hexons isolated from the intracellular pool could differ from those incorporated in the virion [ 5 ] , the identity of tryptic finger-prints of adenovirus type 2 hexon antigen crystallized from infected cell extracts and of hexon polypeptide isolated from mature virus particles implies a structural identity between incorporated and non-incorporated hexons [6].Hexon capsomer is an oligomeric protein, 300000 in molecular weight I n this study, the cross-linking reagent glutaraldehyde [16], has been employed to obtain information regarding the fashion in which the hexon polypeptide subunits interact and the resulting structural and biological changes. To prevent confusion in terms between cross-linked oligomers of complete hexon capsomers and cross-linked oligomers of hexon polypeptide units, these latter were referred to as hexon monomer, dimer, trimer, etc. the terms of capsomer dimer and trimer being reserved to assemblies of several hexon capsomers. MATERIALS AND METHODS Isolation and Purification of Adenovirus HexonsThe method of isolation and purification of adenovirus type2 soluble hexons has been described in details elsewhere [17]. It comprised ammonium sulfate precipitation of an infected cell freon extract, DEAE-Sephadex(A 50) chromatography and chromatography on hydroxyapatite column. The final hexon preparation was homogeneous with regard to the following criteria : electron microscopy, immunoEm.

Temperature-sensitive mutant of adenovirus type 2 blocked in virion assembly: accumulation of light intermediate particles

“…Figure 8 compares the yields of infectious ts1l2 obtained after shift-down with the growth curves at each temperature. Until 18 h p.i., there P V I was little or no effect of the nonpermissive tem-P V11I- 4 perature on the final virus yield. From 30 to 50 -U1 h, the effect of the mutation could not be reversed by a shift-down to a permissive temper-PV11ature.…”

“…served the structure of the tsll2 IM. 4. Sucrose gradient centrifugation analysis of nuclear extracts from WT-and tsll2-infected KB cells.…”

Quantitation of Adenovirus Soluble Antigens by Crossed Immunoelectrophoresis: Application to Serological Characterization of Mutants

“…(ii) It is easier to perform than other serological methods, particularly to assay for free fiber, free penton base and complete penton antigens, (iii) it does not require monospecific antisera against hexon, penton, penton base and free fiber (as in the single radial diffusion technique or in immunoprécipitation of isotopically labeled adenovirus antigens), and all the soluble antigens can be assayed in one single experiment, (iv) It permits us to reveal not only quantitative but also qualitative alterations of the ade novirus ts mutants' structural protein synthesis, e.g., modifications of their electrophoretic mobility or alterations of their antigenic determinants, elicitable in tandem-crossed immunoelectrophoresis [22] with wild-type structural antigens. This method has been successfully employed for analysis of the antigenic determinants of hexon monomer and polymers in cross-linking reactions of hexon capsomers [23], It appears, therefore, to be the method of choice for a rapid immunological screening of adenovirus mutants. …”

The Adenovirus Dodecahedron: Beyond the Platonic Story

“…All adenoviruses are composed of three main capsid proteins or 'capsomers': the hexon, the penton base, and the fiber (Figure 1). The main protein is the hexon, with 240 trimeric capsomers forming the 12 triangular facets of the icosahedral viral capsid [4,5]. The hexon protein is therefore the most abundant structural protein of the adenovirus since 720 hexon copies of the monomer (~110 kDa) are displayed on the virus surface, and it represents 80% of the virus total mass [6].…”