[Probable epitopes of adenovirus capsid proteins]. / Veroiatnye épitopy kapsidnykh belkov adenovirusa.

The hydrophilicity and flexibility patterns of two capsid proteins of type 2 human adenovirus (hexon and fiber) were obtained. Several sites with maximal hydrophilicity and flexibility were revealed; their correlation was partly established. The role of these sites in the formation of adenoviral capsid protein epitopes is discussed.

[Polyacrylamide gel electrophoresis (PAGE) as a method for detecting enteric adenovirus (EAd)].

This paper presents a method which could provide a simple, rapid, economical, and reliable means of detecting or identifying adenoviruses (Advs), rotaviruses (RVs) and reoviruses (ReoVs) in stool suspensions or tissue cultures. The method is based on polyacrylamide gel electrophoresis (PAGE) of the virus nucleic acids, but sample preparation does not need the use of radioactive label, specific DNA probe or antisera. Comparison of the results of PAGE of Adv, RV and ReoV with those of electron microscopy (EM) and/or enzyme-linked immunoabsorbent assay (ELISA) was made. The method is of comparable sensitivity to electron microscopy, and is not limited by amount of specimens obtained, and is thus suitable for application as a batch testing method.

Characterization of the adenovirus 2 virion protein, mu.

Adenovirus 2 virions contain a small, highly basic protein known as mu (mu). Partial sequence analysis of mu labeled with radioactive amino acids showed that it is derived from an 11-kDa virion precursor protein, L2-79R. Amino acid analysis, direct microsequence analysis, time-of-flight mass spectrometer analysis, and chemical synthesis demonstrated that mu is the unmodified, 19 amino acid peptide obtained from the 79-residue precursor by adenovirus-encoded proteinase-mediated cleavage after glycine31 and glycine50. Mu bound tightly to DNA and was located in the virion core. In vitro, mu could precipitate DNA fragments, suggesting that it may have a role in viral chromosome condensation.

Adenovirus type 7 infections in children in New South Wales, Australia.

This study describes our experience of adenovirus type 7 infection in children in New South Wales, Australia. Some aspects of the epidemiology of the infection are also recorded. A community outbreak, a hospital-ward outbreak, sporadic cases, and data from a centralised registry are described. Results of genome-type identification using restriction endonucleases are given. Adenovirus type 7b infection was associated with significant mortality and points to the need, previously expressed, for an adequate vaccine for high-risk infants. Continued surveillance of adenovirus type 7 infections worldwide is necessary to identify genome types so that appropriate vaccines can be developed.

Detection of viral antigens in patients with IgA nephropathy.

Immunofluorescent analysis of viral antigens in the cultured fibroblasts and renal tissues in patients with IgA nephropathy was described. Freeze and thawed extracts of pharyngeal cells obtained from patients with IgA nephropathy, chronic proliferative glomerulonephritis without IgA deposition (PGN) and healthy adults were cultured with human fibroblasts, i.e. Hel cells, with or without addition of 5-iodine 2'-deoxy-uridine (IUDR) at 37 degrees C for 2 weeks. These fibroblasts and renal sections were stained with several kinds of FITC-labeled antiviral antibodies. Deposition of adeno, herpes simplex, varicella zoster or parainfluenza 3 was observed not only in the renal sections but also in the nuclear regions and/or cytoplasm of Hel cells after incubation of extracts of pharyngeal cells with or without IUDR from patients with IgA nephropathy. It is indicated that antigenic stimulation in the upper respiratory tracts may be due to several different types of DNA and/or RNA viruses in patients with IgA nephropathy. It appears that these antigenic substances show some heterogeneity among these patients.

Phosphorylation at serine 89 induces a shift in gel mobility but has little effect on the function of adenovirus type 5 E1A proteins.

Phosphorylation at serine 89 was shown to be the major cause of the shift in gel migration of the 289R and 243R early region 1A (E1A) proteins of human adenovirus type 5. However, conversion of Ser-89 to alanine by site-directed mutagenesis did not abolish E1A transactivating or transforming activities, suggesting that phosphorylation at this site is not necessary for these E1A functions.

The composed antigenic structure of the adenovirus hexon protein.

Three panels of MAbs were prepared against AV1, AV35, and BAV2 hexons, respectively. It was shown, that in all cases, antibodies are developed against the genus specific determinant of the adenovirus hexon. The other MAbs specified numerous identical or overlapping epitopes on the hexon types studied. The epitopes could be characterized as intertype-, intersubgenus-, subgenus- and type-specific ones beside the genus-specific determinant based on the RPs of the MAbs from indirect ELISA and passive HA results. The identical epitopes are present in more than one copy on the trimeric form of the hexon capsomer. The epitopes on the hexon molecule could be separated into three antigenic sites, of which one antigenic site is characterized by seven epitope clusters (antigenic site II). Monoclonal antibodies were able to precipitate different hexon types in gel diffusion tests by which the differentiation of the distinct epitopes seemed to be possible. With the help of monoclonal antibodies to AV1 hexon, 17 hours after the infection, hexons (or epitopes) were detected in the cytoplasm and in the nucleus of the infected cells showing different distribution patterns in indirect immunofluorescence assay.

Psoralen-cross-linking study of the organization of intracellular adenovirus nucleoprotein complexes.

We used the psoralen-cross-linking technique to investigate the structures of adenovirus nucleoprotein complexes during infection. At late times after infection, three types of psoralen cross-linking patterns were observed. A high cross-linking pattern (type I), with about one cross-link in every 10 to 17 base pairs, was found for the newly synthesized and the bulk of the adenovirus late chromatin. Viral templates involved in replication, transcription, and recombination were all found to exhibit this cross-linking pattern. These results suggest that there is no nucleosome-like organization in the unpackaged late adenovirus nucleoprotein complexes. The second type of cross-linking pattern (type II) had a low cross-linking density of about one cross-link in every 700 to 1,000 base pairs. This cross-linking pattern was found to be associated with the viral DNA in the mature virus particles. The sequences at the termini of the virion DNAs, however, were found to have higher cross-linking densities, as shown by electron microscopy. The third type of cross-linking pattern (type III) was composed of a mixture of various proportions of type I and type II patterns in a single molecule. This mixed cross-linking pattern suggests that these molecules are virion assembly intermediates, with viral DNA being partially packaged in the virus particles. The organization of adenovirus nucleoprotein complexes at early times after infection was analyzed by the gel electrophoresis technique following digestion of the DNA with a restriction enzyme that was inhibited by cross-links. Our data suggest that the viral nucleoprotein complexes at early times after infection have accessibility to psoralen cross-linking between the virion DNA and the late viral nucleoprotein complexes. The observed cross-linking density of the early nucleoprotein complex DNA, however, was inconsistent with the nucleosomelike organization suggested by previous investigators.

Adenovirus transcriptional complexes contain EIa encoded tumour antigens physically bound to cellular proteins.

Adenovirus type 12 transcriptional complexes were isolated from cells during the early phase of infection. Sedimentation analysis identified a fast sedimenting complex type I and a slow sedimenting complex type II. Both complexes made virus specific RNA complementary to all the early genes and both contained viral DNA, which in type II but not in type I had nucleosome like configuration. Analysis of the proteins of the complexes with antiserum against Ad 12 EIa-beta-galactosidase fusion protein expressed in E. coli demonstrated the following: (a) type I complex contained EIa 45 K protein, which co-precipitated with cellular proteins of mol. wt. 42, 58, and 60 K, (b) type II complex contained EIa 47 K protein, which co-precipitated with major cellular proteins of 35, 40-46 K and minor proteins of 58, 60, 68, 76, 86, and 120-150 K. Association of EIa specific and cellular proteins to transcriptional complexes was sensitive to both 1 M NaCl and DNAse I indicating the DNA binding nature of these proteins. Treatment of transcriptional complexes with 1 M NaCl or DNase I released EIa proteins, which still remained strongly bound to cellular proteins. These findings suggested that EIa proteins bind to viral DNA and that this binding is probably mediated by cellular proteins.

Identification of adenovirus type 2 early region 1B proteins that share the same amino terminus as do the 495R and 155R proteins.

Adenovirus type 2 early region 1B (E1B) proteins synthesized in vitro were fractionated chromatographically and characterized by peptide and sequence analysis and by reaction with peptide-specific antisera targeted to either the N or C terminus of either of two overlapping E1B reading frames (175 or 495 codons). In addition to the previously identified E1B-495R, E1B-175R, and E1B-155R species, two other E1B proteins of similar electrophoretic mobility to the 175R protein were identified. E1B-82R is an abundant product in vitro and in vivo that has the same N terminus as that of the 495R and 155R proteins but a different C terminus. The structure of 82R is predicted by the structure of the abundant 13S (1.02-kilobase) E1B mRNA. E1B-168R is a novel minor species consisting of the 24 amino-terminal residues of the 495R protein fused to the entire polypeptide IX sequence. An additional, minor 16,000-molecular-weight polypeptide was detected that may correspond to a predicted 92R E1B protein, but definitive identification was not possible. These observations establish that the leftmost portion (78 codons) of the 495-codon reading frame, which overlaps the right half of the 175-codon reading frame, is expressed as an abundant protein that does not contain other 495R sequences. This region, which may participate in the regulation of region E1A expression, may thus constitute a functional domain distinct from the rightward portion of the 495R protein.

Crystallization, enzymatic cleavage, and the polarity of the adenovirus type 2 fiber.

Crystals of the fiber protein of adenovirus type 2 have been grown. Analysis of these crystals (type I crystals) showed that they were composed of fiber polypeptide with a lower apparent molecular weight (60 kDa) than that of the soluble or virion-incorporated fiber (62 kDa). N-terminal sequencing revealed that the fiber polypeptide chain of 60 kDa was cleaved at tyrosine17 from the N-end. The C-terminus remained intact. Assays with protease inhibitors suggested that the spontaneous cleavage of the fiber occurring upon its crystallization was due to a cellular, calcium-dependent, chymotrypsin-like protease co-purifying with the fiber and activated during hydroxyapatite chromatography. Crystallization of fiber purified in the presence of chymostatin provided crystals of a different structure under the electron microscope (crystals of type II), composed of 62-kDa fiber polypeptide units. The 62-kDa fiber from the type II crystals, as well as the 62-kDa fiber isolated from infected cell extracts, were able to associate with the penton base in vitro to form a penton capsomer. The 60-kDa fiber has lost this capacity. The accessibility of the N- and C-termini of the fiber inside the penton structure was probed by anti-peptide sera after limited proteolysis. The results are consistent with a polarity of the fiber in which its N-terminus is oriented toward the penton base, the C-terminal domain corresponding to the distal knob.

Isolation and characterization of adenovirus core nucleoprotein subunits.

Digestion of adenovirus type 2 (Ad2) or Ad5 cores with micrococcal nuclease generated four nucleoprotein species that could be resolved by electrophoresis in low-ionic-strength polyacrylamide gels: these nucleoproteins displayed mobilities equivalent to those of DNA fragments of 900 to 1,025, 775 to 850, 650 to 725, and 525 to 600 base pairs (bp) and thus were readily distinguishable from HeLa cell mononucleosomes. The DNA fragments associated with the core nucleoprotein species were more than 250 to 90 bp long. Nucleoproteins containing 150, 120, or 90 bp of DNA were the most stable. Polypeptide VII was associated with each of the nucleoprotein species liberated from Ad2 cores. These data suggest that polypeptide VII and viral DNA of 90 to 150 bp comprise the unit particle of the Ad2 or Ad5 core nucleoproteins.

Column purification of adenovirus cores.

A simple and efficient technique for purifying adenoviral chromatin (nucleoprotein cores) with Sephacryl S-1000 is described. This method is significantly faster than previous methods and gives a higher degree of purity with an increased recovery of the nucleoprotein. In addition, the structural integrity of the cores is maintained.

Analysis of retinoblastoma for human adenovirus and human JC virus genome integration.

Human adenovirus groups A and B have an oncogenic potential in newborn rodents. Especially, adenovirus type 12 is known to induce retinoblastoma-like tumor in baboons and transform human embryo retinoblast cells in vitro. Human JC virus is also known to produce a variety of tumors in newborn rodents, including retinoblastoma-like tumor. In this experiment, cell DNAs of human retinoblastomas were assayed for each of the transforming gene sequences of adenovirus groups A, B, C, D and E and for JC virus gene sequences, by using Southern blot hybridization. None of these viral gene sequences were detected at the level of 0.1-0.5 copy per diploid cell DNA in all of 11 retinoblastomas, including 6 retinoblastomas previously examined for adenovirus 12-transforming gene sequences. This led to a conclusion that most, if not all, adenoviruses and JC virus play no essential role in the etiology of human retinoblastoma, although there were experimental models of retinoblastoma induced by these viruses.

Gastroenteritis in infants, associated with a genome type of adenovirus 31 and with combined rotavirus and adenovirus 31 infection.

In an infants' ward, gastroenteritis occurred in five children in two groups, probably by nosocomial spread of adenovirus 31 (three cases) and adenovirus 31 + rotavirus (two cases). The infants recovered well. The DNA of adenovirus 31 isolates was analysed with ten restriction endonucleases and found identical for all five strains, but different from the prototype.

[Stability of the structure and antigenic determinants of adenovirus type 1 native hexon to proteases]. / Ustoichivost' struktury i antigennykh determinantov nativnogo geksonaadenovirusa tipa 1 k proteazam.

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.

Genome analysis of species 3 adenoviruses isolated during summer outbreaks of conjunctivitis and pharyngoconjunctival fever in the Glasgow and London areas in 1981.

Genome analysis was performed on 125 adenovirus isolates from conjunctival swabs of patients with conjunctivitis obtained in Glasgow between 1981 and 1984. A summer outbreak in 1981 was mainly due to species 3 adenoviruses, of which genotype 3GB and five different genotypic variants cocirculated. Three species 3 variants were also observed in 1982. The genome changes of variants were located on physical maps of the Ad3 reference strain and found to be clustered near the ends of the adenovirus DNA (including the fiber area), whereas the hexon coding region was unaltered. In contrast to the genome heterogeneity observed among the species 3 adenoviruses collected in Glasgow in 1981 it was found that all 69 Ad3 isolates obtained from an outbreak of pharyngoconjunctival fever in a boarding school near London during the summer of 1981 possessed the 3GB genotype.

Antigenic homogeneity of adenovirus types 1, 2, 5, and 6.

One hundred twenty strains of adenovirus types 1, 2, 5, and 6 (subgenus C), isolated at various times and places, were tested by neutralization and hemagglutination inhibition with two antisera of each type. Except for one type 5 strain they reacted largely type-specifically in both tests and neither qualitative nor quantitative differences were observed. Serologically intermediate strains were not found. The antigenic homogeneity is in contrast to the heterogeneity of the genomes of subgenus C adenoviruses.

Studies on fastidious adenoviruses in Ontario: a distinct strain associated with gastroenteritis.

From 100 cases of gastroenteritis among children caused by adenovirus infection in Ontario, 33 virus isolates were divided into three categories according to their biological behavior in tissue cultures. So far, the results of neutralization tests, structural protein analysis, and DNA restriction patterns showed that the virus of category 1 was similar to adenovirus type 40. However, the adenovirus of category 2 was a distinct adenovirus which shared some similarities with adenovirus type 5. Viruses of category 3 are still under investigation.