The 100K-chaperone protein from adenovirus serotype 2 (Subgroup C) assists in trimerization and nuclear localization of hexons from subgroups C and B adenoviruses

Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.     

Antigenic determinants of adenovirus capsids. II. Homogeneity of hexons, and accessibility of their determinants, in the virion.

We have tested the two principal theories which explain the previous finding that small amounts of type-specific antibody to the adenovirus hexon can neutralize infectivity, whereas even large amounts of cross-reactive antibody do not. a) It has been suggested that the type-specific determinants are especially prominent in the virion. We have therefore measured the capacity of whole virus to bind appropriate antibodies, using a sensitive radioimmunoprecipitation (RIP) system. In fact, virions bound type-specific and cross-reactive antibodies impartially. Moreover, they bound both much less effectively than did free hexon or disrupted virus, suggesting that many of each kind of determinant are inaccessible in virions. b) It has been suggested that the type-specific determinants are confined to those hexons located next to the pentons, and that they are the targets for neutralizing antibody. We have therefore studied the antigenicity of peripentonal and nonamer hexons isolated from virions, and found that each possessed both kinds of determinants. Furthermore, these were present in the same proportion as in hexons purified from the soluble antigens in infected cells ("free hexons"). We concluded that the mechanism of neutralization by antibody is complicated, and that the type-specific determinants exposed on the virion must play a crucial role.     

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.     

Immunological Relationships Between Hexons of Certain Human Adenoviruses

Antisera against hexons of serotypes 2, 4, 5, and 6 (subgroup III), and 15 (subgroup II) were absorbed with purified hexons of various serotypes representing the different subgroups of human adenoviruses. Group, subgroup, and type specificities of hexons could be distinguished. The subgroup specificity of type 4 hexons resembled that of hexons of subgroup I members (types 3, 11, and 16). Antihexon sera gave a type-specific inhibition of virion-associated hemagglutinin. The inhibiting activity of different sera was found to be inversely related to the length of fibers of the serotype concerned. Virions of serotypes carrying fibers shorter than about 20 nm (types 3, 4, 9, 11, and 15) were readily inhibited, whereas those of serotypes with longer fibers (types 2 and 6) were inhibited only by relatively large amounts of antibody measured in terms of homotypic complement fixation activity. The reciprocal cross-neutralization between serotypes 4 and 16 was studied separately. Hexons of both serotypes each carried a type-specific component and, in addition, a unique antigen specificity common to the two types. This common antigen specificity was interpreted to be available to a larger extent at the surface of virions (and probably also isolated hexons) of type 4 than of type 16. These results suggest an explanation for the predominantly one-sided character of the cross-neutralization between types 4 and 16.     

In vitro reconstitution, hexon bonding and handedness of incomplete adenovirus capsid

Purified groups of nine hexons (nonamers) from trypsin and sodium deoxycholate-disrupted adenovirus type 5 were found to re-aggregate at low pH values into pairs, rings of five and icosahedral shells of twenty nonamers. Electron microscopy and ultracentrifugation showed that these shells are the same as normal adenovirus capsids minus the twelve vertex assemblies of six capsomers each.The pyramidal rings of five nonamers provided the first clear evidence that the adenovirus capsid is left-handed. Dissociation of the nonamers suggested that they are organized as a central hexon trimer plus three differently bonded dimers, and not as a true p3 net. New evidence is presented, based on two-dimensional hexon crystals, that individual hexons have 3-fold cyclic symmetry and the whole question of hexon-hexon bonding is discussed in the light of these observations.     

Antigenic composition of adenovirus hexons]

Quantitative relations between the group-specific and the type-specific components of the hexons of adenovirus type 2 and 5 were studied by means of FITC-conjugated Fab-fragments of antibodies directed against type 2 and type 5 hexons. From the sedimentation constant of the complexes of hexons and Fab in the region of excess of Fab we conclude that there are at least 20 determinants on the hexon. Half of these are type-specific and the others are group-specific. Both components of the type 2 hexon consist of equal parts of carbodiimide sensitive and carbodiimide resistent determinants.     

Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein.

Autotaxin (ATX) is a potent human motility-stimulating protein that has been identified in the conditioned medium from A2058 melanoma cells. This protein has been purified to homogeneity utilizing a strategy involving five column steps. Homogeneity of ATX was verified by two-dimensional gel electrophoresis. The molecular size of ATX is 125 kDa, and it has an isoelectric point of 7.7 +/- 0.2. Purified ATX was digested with cyanogen bromide and trypsin, and the resulting ATX peptides were purified by reverse-phase high performance liquid chromatography. Eleven peptides were subjected to amino acid sequence analysis, and 114 residues were identified. The partial amino acid sequences and the amino acid composition obtained for ATX show that it does not exhibit any significant homology to known growth factors or previously described motility factors. At picomolar concentrations, ATX stimulates both random and directed migration of human A2058 melanoma cells. Pretreatment of the melanoma cells with pertussis toxin abolishes the response to purified ATX, indicating that ATX stimulates motility through a receptor acting via a pertussis toxin-sensitive G protein.     

An immunochemical aid to sequence determination of proteins.

A specific radioimmunoassay for peptides has been developed using ¹²⁵I-labeled peptides and a double-antibody precipitation. Cross-reacting peptides are measured by inhibition of the binding of the labeled cyanogen bromide peptide to its antibody. The assay, which allows detection of picomole quantities, was used to monitor the purification of two overlapping tryptic peptides from a complex mixture of peptides. These were shown to contain a portion of the sequence of the radio-labeled cyanogen bromide peptide and a portion of the sequence of a cyanogen bromide peptide which follows in the polypeptide chain. The need to analyze many fractions in a digest in order to locate a desired peptide is thus avoided. The general suitability of this method for the purification of specific peptides from digestion mixtures of other large proteins is discussed.     

Primary structure of the elongation factor G from Escherichia coli. VI. Structure of peptides of cyanogen bromide cleavage of the G-factor molecule

Peptides obtained as a result of cyanogen bromide cleavage of the G-factor have been studied. All 12 peptides embracing the whole structure of fragment T4 have been isolated. For their amino acid sequence determination, cyanogen bromide peptides have been further cleaved with trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease and BNPS-skatole. The complete primary structure of 9 from 12 cyanogen bromide peptides has been determined.     

Thf complete amino acid sequence of C-I (apoLp-Ser), an apolipoprotein from human very low density lipoproteins.

C-I was prepared from very low density lipoproteins of patients with familial type V hyperliporproteinemia. Peptides from tryptic digests of unmodified and succinylated C-I, chymotryptic peptides, and the products of cayanogen bromide cleavage were isolated and characterized. Sequence analysis of tryptic peptides was performed by the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) technique and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. Alignment of the tryptic fragments within the cyanogen bromide and succinyl-tryptic peptides was confirmed by the overlap chymotryptic peptides. The complete amino acid sequence of C-I, 57 residues in length, does not reveal any obvious basis for its lipophilic properties.     

Quantitation of types I and III collagens in human tissue samples and cell culture by cyanogen bromide peptide analysis

A procedure for the quantitation of types I and III collagens by cyanogen bromide peptide analysis was developed with the aim of eliminating certain problems associated with this method. Ion-exchange chromatography reduced high background levels on gel scans used to quantitate the peptides; reduction with beta-mercaptoethanol substantially increased the efficiency of the cyanogen bromide cleavage; use of a concave gradient in acrylamide from 8 to 20% improved the resolution of cyanogen bromide peptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and a normalization procedure eliminated variations due to differences in the amount of material loaded on the gel system. This method of quantitation was applied to human aorta samples and to collagen secreted by human skin fibroblasts. Metachromasy of type I and type III collagen cyanogen bromide peptides stained with Coomassie blue R-250 was established and this was used as an index of the purity of the cyanogen bromide peptide preparations. Type I and III collagens were prepared from human placental tissue, and these purified collagens were used to construct calibration curves to determine the relationship between the quantity of diagnostic cyanogen bromide peptides present and the composition of the sample in terms of types I and III collagens.     

Selective dansylation of M protein within intact influenza virions.

Exposure of purified influenza virions to [14C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.     

Adenovirus type 2 terminal protein: purification and comparison of tryptic peptides with known adenovirus-coded proteins.

The protein covalently bound to the 5' termini of adenovirus type 2 DNA has been purified from virus labeled with [35S]methionine, using exclusion chromatography of disrupted virions to isolate the DNA-protein complex, which is then digested with DNase. The terminal protein isolated from mature virus is most effectively labeled if the cells are exposed to [35S]methionine during the "intermediate" period of 13 to 21 h postinfection, suggesting that the protein is synthesized during this interval. The tryptic peptides of the terminal protein were compared with those of several known adenovirus-coded proteins and found to be unrelated. In particular, the terminal protein is not related to the 38-50K early proteins encoded by the leftmost 4.4% of the adenovirus genome, one region essential for the transforming activity of the virus. Neither is it related to the 72K single-strand-specific DNA binding protein, the minor virion component IVa2, or the major capsid component hexon.     

Peptide analysis by isoelectric focusing in polyacrylamide gels

We have examined the use of isoelectric focusing in polyacrylamide gels for the analysis of heterogeneous mixtures of cyanogen bromide peptides. High resolution, sensitivity, and reproducibility are obtainable under conditions which are described. Peptides having molecular weights above 1000 or 2000 can be visualized by fixation and staining. The presence of urea in the gels is important to the procedure; formation of carbamylated derivatives from this cause occurs at most in trace amounts in unfavorable cases. No artifactual heterogenelty from any other cause was apparent.     

Structural and immunochemical analysis of the products of proteolysis of simian adenovirus hexons

Hexon capsomers of simian adenovirus sim16 (SA7) and of human adenoviruses h5 (Ad5) and h6 (Ad6) were proteolytically digested and the resulting products studied by SDS-polyacrylamide gel electrophoresis and by radioimmunoprecipitation analysis. The trypsinolysis of native SA7 hexon leads to a stable molecular "core" containing 4-5 fragment species of 10 to 65 kDa and resembling the intact capsomer in quarternary structure (trimer). Similar cores but consisting of smaller fragments (less than 40 kDa) were obtained after chymotryptic digestion of native SA7, Ad5 and Ad6 hexons. The chymotryptic hexon fragments were also held together in pseudotrimeric structures. The similarity of proteolytic hexon fragment patterns between different primate adenoviral hexons suggested a homology to exist in localisation of the exposed tryptic and chymotryptic cleavage sites in their respective hexon polypeptide chains. Papain caused a complete hydrolysis of native SA7 hexon (trimer) yielding small peptides, but at first stage of digestion a stable papain hexon core containing small fragments (less than 10 kDa) was observed. The tryptic SA7 hexon cores in native state retained their antigenicity in reactions with homo- and heterologous antibodies, but after core denaturation the resulting fragments had no antigenic activity of native capsomer. In contrast to the data previously published, chymotryptic cores of SA7, Ad5 and Ad6 hexons not only reacted with respective homologous antibodies but also retained (at least in part) cross-reactive antigenic determinants. The questions of formation and stability of native adenoviral hexon conformation are discussed as well as the possible nature of hexon antigenic determinants.     

Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic,molecular modeling,and sequence-based methods

A major impediment to the use of adenovirus as a gene therapy vector and for vaccine applications is the host immune response to adenovirus hexon-the major protein component of the icosahedral capsid. A solution may lie in novel vectors with modified or chimeric hexons designed to evade the immune response. To facilitate this approach, we have distinguished the portion of hexon that all serotypes have in common from the hypervariable regions that are responsible for capsid diversity and type-specific immunogenicity. The common hexon core-conserved because it forms the viral capsid-sets boundaries to the regions where modifications can be made to produce nonnative hexons. The core has been defined from the large and diverse set of known hexon sequences by an accurate alignment based on the newly refined crystal structures of human adenovirus types 2 (Ad2) and Ad5 hexon. Comparison of the two hexon models, which are the most accurate so far, reveals that over 90% of the residues in each have three-dimensional positions that closely match. Structures for more distant hexons were predicted by building molecular models of human Ad4, chimpanzee adenovirus (AdC68), and fowl adenovirus 1 (FAV1 or CELO). The five structures were then used to guide the alignment of the 40 full-length (>900 residues) hexon sequences in public databases. Distance- and parsimony-based phylogenetic trees are consistent and reveal evolutionary relationships between adenovirus types that parallel those of their animal hosts. The combination of crystallography, molecular modeling, and phylogenetic analysis defines a conserved molecular core that can serve as the armature for the directed design of novel hexons.     

Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides

Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha. Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305--316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120, 279--287], the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alteration of alpha helices and beta structure. The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed.     

Hexagonal crystalline arrays of adenovirus type 1 hexons

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.     

Two- and three-dimensional hexon crystals.

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.     

Pseudopackaging of adenovirus type 5 genomes into capsids containing the hexon proteins of adenovirus serotypes B, D, or E

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive (ts) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temperature, Ad5 ts147 replicates normally, providing a source of Ad5 DNA for virus assembly, but does not produce virus particles due to the hexon protein mutation. Coinfection of Ad5 ts147 with a wild-type virus of other Ad serotypes (Ad3, Ad4, or Ad9), which supply functional hexon proteins, resulted in the pseudopackaging of the Ad5 DNA genome. Furthermore, the pseudopackaged Ad5 DNA virions obtained in the coinfections were infectious. Therefore, switching hexons did not impair the infectivity or uncoating process of the pseudopackaged virion. Since hexon protein is a major antigenic determinant of the Ad capsid, this approach may prove useful to reduce the antigenicity of therapeutic Ad vectors and allow repeated vector administration.