Adenovirus Hexon Protein Research Articles

Detection of Adenovirus in the waters of the Seine River estuary by nested-PCR

Several systems for isolating viruses from environmental samples have been tested. The most promising method is based on genomic amplification. The authors attempted to detect adenovirus in nucleic-acid extracts from the Seine River estuary by a two-step amplification of a 220–bp segment of the conserved coding region of type 2 adenovirus hexon protein L3. The primers used in this study detected the most prevalent adenovirus serotypes in human disease in France, but not other virus strains or bacteria. The sensitivity of the nested polymerase chain reaction (PCR) amplification was estimated to be 102copies of the adenovirus target sequence per ml of Seine River water. Nucleic-acid extracts from Seine River estuary waters were analysed and some tested positive for the presence of adenoviruses.

Adenovirus mixture isolated from the brain of an AIDS patient with encephalitis

A mixture of adenoviruses 31 and 49 was isolated from the brain of an AIDS patient with encephalitis. Adenovirus hexon protein was detected in neurons by indirect immunofluorescence. By restriction endonuclease analysis both adenovirus 31 and 49 were shown to be new genotypes. This is the first report of the isolation of a mixture of adenoviruses from adenovirus encephalitis and the first association of adenovirus 49, a new candidate serotype, with encephalitis.

Purification of adenovirus hexon protein by high-performance liquid chromatography

Adenovirus (Type 2)-infected Hella cells were sonicated and treated with 1,1,2-trichlorotrifluoroethane. The water-soluble extract was ultracentrifuged and the supernantant, containing the dissociated proteins, was subjected to anion-exchange high-performance liquid chromatography on a Mono Q column in 50 m M bis-TrisHCl (pH 6.5). Elution with a linear salt gradient resulted in a major peak, containing the hexon protein.

The synthesis and intracellular localization of adenovirus hexon protein studied by microinjection of mRNA into human cells

Polyadenylated RNA isolated 18 h after infection of HeLa cells with adenovirus type 2 was both translated in vitro and microinjected into the cytoplasm of human transformed amnion cells. The hexon polypeptide could be specifically immunoprecipitated from the products of cell-free translation with a rabbit-anti-hexon serum. The same serum when used in immunofluorescence assays of microinjected cells revealed hexon protein synthesized 6 h after microinjection. The intensity of the staining persisted up to 16 h after injection of messenger RNA (mRNA). Newly-synthesized hexon protein was characteristically located mainly in the nucleus. Essentially the same results were obtained when normal amnion cells were microinjected.

The adenovirus hexon protein. The primary structure of the polypeptide and its correlation with the hexon gene.

The primary structure of the adenovirus hexon polypeptide has been determined by amino acid sequence studies of peptides from all regions of the molecule combined with sequence analysis of selected areas of its gene. The sequence presented contains 966 unique amino acid residues. Overlapping peptides recovered from CNBr cleavage and from digestions with proteolytic enzymes were analyzed, as well as DNA segments around sites for restriction endonucleases in the hexon gene. The primary structure is in good agreement with the total composition of the protein, with the compositions of individual CNBr fragments, and with known locations of restriction enzyme cleavage sites in the gene. Distinct regions of internal homology do not occur in the structure. The entire hexon polypeptide is encoded by a contiguous DNA sequence without intervening sequences.

Order of the CNBr fragments in the adenovirus hexon protein.

Order of the CNBr fragments in the adenovirus hexon protein.

Fractionation of CNBr fragments and primary structures of peptides of the adenovirus hexon protein.

The adenovirus hexon protein has been carboxymethylated with 14C-labeled iodoacetate. After treatment with CNBr, the peptide mixture was fractionated into fragments with seven size classes by exclusion chromatography. Large fragments were further purified by CM-cellulose chromatography in urea, and small fragments were purified by high voltage paper electrophoresis. Amino acid sequences of pure fragments have been determined by combined use of sequenator-based direct degradations, and of manual t-dimethylaminonaphthalene-1-sulfonyl-monitored Edman degradations subsequent to enzymatic redigestions. Fractionations are given, the primary structures of 22 CNBr fragments containing a total of 677 residues are reported, and the analytical aspects of the structural properties are considered.

Structural analysis of four large CNBr fragments from [14C]-carboxymethylated adenovirus hexon protein.

Structural analysis of four large CNBr fragments from [14C]-carboxymethylated adenovirus hexon protein.

Limited proteolysis and a reactive cysteine residue define accesible regions in the native conformation of the adenovirus hexon protein.

The sulfhydryl group of one cysteine residue in the adenovirus hexon protein is accessible to alkylation in the absence of denaturating agents. This residue was [14C]carboxymethylated and characterized in peptides after proteolytic treatments. It was identified in two different types of digest and corresponded to the cysteine residue most accessible to oxidation. It is located close to residue 670 in the tentative amino acid sequence of the entire polypeptide. Trypsin has a highly limited action on the native hexon protein. Only three peptide bonds are fully susceptible to cleavage. These were found to occur in the N-terminal third of the protein, and were characterized as cleavages close to positions 150, 180 and 320 in the tentative structure of the polypeptide. Trypsin therefore liberates four fragments in major yield, with approximate Mr of 3000, 15000, 17000 and 78000. The smallest fragment corresponds to a highly acidic region. A limited, but less restricted susceptibility of hexon to chymotryptic cleavage was also found, and four major sites were characterized. The cysteine labelling and the proteolytic treatments define accessible regions in the native conformation of hexon. Together with previously assigned residue distributions, they suggest the subdivision of the entire polypeptide chain into three approximately equal parts, with accessible sites in the border regions, as well as in the middle of at least the N-terminal third. One model compatible with all data is presented.

Radioactive labelling of acidic regions in the adenovirus hexon protein through metabolic conversion of [ 14C] acetate

Radioactive labelling of acidic regions in the adenovirus hexon protein through metabolic conversion of [ 14C] acetate