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.     

Small angle x-ray scattering studies on adenovirus type 2 hexon

Adenovirus type 2 hexons have been studied in solution by small angle X-ray scattering, and the following molecular parameters determined: radius of gyration (Rg) = 4.9 nm, molecular weight (M) = 310.000, invariant volume (Vinv) = 630 mn3, maximal distance (Dmax) = 14.5--15.5 nm. A diffraction pattern was obtained up to an angular increment of h = 2.5 nm-1. Various models for the hexon have been explored by calculating the diffraction pattern from the Debye formula for 1200 spheres arranged to define the scattering volume of each model. Models were first built according to electron micrographic results. Later, preliminary results of a crystallographic study were used for model building. The experimental pattern and the pattern resulting from the model determined by crystallographic methods were compared and showed good agreement.     

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.     

Diffusion coefficient and molecular weight of type 5 adenovirus by photon-correlation spectroscopy

The technique of photon-correlation spectroscopy (intensity fluctuation spectroscopy) is applied to light scattered from type 5 adenovirus undergoing Brownian motion in solution and the translation diffusion coefficient (D₂₀,W) measured to be 0.367 ± 0.003 Fick units. Using Svedberg's equation with previously determined parameters, a molecular weight of 165 · 10⁶ ± 5 · 10⁶ is obtained.     

A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA.Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to α-amanitin. In isolated nuclei, both RNA species are labeled with β-³²P-labeled GTP, which suggests that they are initiated at separate promoter sites.     

Stability of the structure and antigenic determinants of adenovirus type 1 native hexon to proteases

Hexon capsomers of human adenovirus type 1 (h1) labeled by iodine 125 were digested in a native state (trimers) by trypsin, chymotrypsin or papain, and the resulting hydrolysates were analyzed by SDS-PAGE. In each case, a discrete and temporally stable pattern of relatively large fragments was revealed. The degree of hexon polypeptide hydrolysis was maximal for papain, intermediate for chymotrypsin and minimal for trypsin, the largest fragments in the digest being 32, 40 and 80 kD, respectively. At room temperature, all the electrophoretically discernible hexon proteolytical fragments were held together in structures resembling intact hexon trimers and could be regarded as "hexon cores", of which papain hexon cores were the most stable during SDS-PAGE. Radioimmunoprecipitation analysis revealed a complete absence of native hexon antigenicity in thermodenaturated fragments of hexon protease digests, while native trypsin, chymotrypsin and papain hexon cores could be precipitated by hexon-specific antibodies. The immunoprecipitated material contained all of the hexon fragments found in appropriate hexon cores and retained the structure of the original cores. Trypsin, chymotrypsin and papain hexon cores were shown to possess at least part of native Ad h1 hexon antigenic determinants of each of the following specificities: species-specific (epsilon), cross-reactive with hexon of human adenoviruses (h3 and h6), simian adenovirus (sim 16), bovine adenoviruses (bos 3 and bos 7) and avian adenovirus (Aviadenovirus gal 1 or CELO). Thus, the full spectrum of known hexon antigenic determinants (species-specific to intergenus-crossreactive) is at least portly stable against protease attack of native hexon capsomers.     

Antibodies with specificities against a dispase-produced 15-kilodalton hexon fragment neutralize adenovirus type 2 infectivity.

During the entrance of adenovirus type 2 into cells, it has been suggested that the virion undergoes a conformational change. In this investigation, we have further characterized the hypothetical conformational change, which the structural protein hexon undergoes in response to low pH. From pH 5.0 to pH 6.0, the proteolytic enzyme dispase cleaved the hexon into a few distinct fragments with a dominating low-molecular-weight fragment with a molecular weight of 15,000 (15K peptide), whereas between pH 6.5 and pH 8.0, the cleavage of the hexon was negligible. The degradation of the hexon with dispase at low pH was not due to an increased activity or alteration of the active site of dispase at low pH. The 15K fragment was identified as a segment of the N-terminal part of the hexon polypeptide beginning at amino acid residue 5. An immune serum produced in response to acid-treated and glutaraldehyde-fixed hexons contained a small amount of antibodies directed towards the 15K fragment, as judged by Western immunoblotting. An anti-15K antibody fraction was isolated by affinity chromatography by removing antibodies recognizing the hexon in the alkaline configuration. Such antibodies displayed a higher relative titer at pH 5.0 than at pH 7.5 in an enzyme-linked immunosorbent assay. The isolated antibodies showed a specific neutralizing capacity five times higher than that of the corresponding unfractionated polyclonal anti-hexon serum; however, the neutralizing ability was independent of pH. The neutralization of adenovirus type 2 infection by the isolated anti-15K antibodies implies that the N-terminal end of the hexon may play a critical role in the early steps of the virion-cell interaction.     

pH-Dependent exposure of endoproteolytic cleavage sites of the adenovirus 2 hexon protein

Abstract Physiological ionic strength conditions prevented low pH-mediated destabilization of the adenovirion. A conformational change of the virion was induced at low pH as demonstrated by endoproteolytic cleavage of virions with dispase at pH 5.0. Hidden cleavage sites of the hexons were exposed and upon enzymatic digestion, virions still were intact as physical entities. Enzymatic cleavage of the hexon protein increased its hydrophobicity.     

The low molecular weight of RNAs of adenovirus 2-infected cells

The cytoplasm of HeLa cells infected with adenovirus type 2 contains many species of low molecular weight RNA, including several of viral origin. In addition to a 9 S messenger RNA, the viral genome gives rise to two species of virus-associated RNA: the major species is 5.5 S RNA or virus-associated RNA_I, and the minor species is 5.2 S RNA or virus-associated RNA_II. Virus-associated RNA_I occurs in the cytoplasm in several electrophoretically separable forms, and its sequences are also present in high molecular weight nuclear RNA but not in cytoplasmic mRNA. The structure of virus-associated RNA_II is shown to be distinct from that of the major species, and the position of its gene is mapped on the viral genome. The two virus-associated RNA genes are located on the r strand near position 30 of the adenovirus type 2 physical map, and are separated by a spacer of about 75 base-pairs.