[Structural and immunochemical analysis of the products of proteolysis of simian adenovirus hexons]. / Strukturnyi i immunokhimicheskii analiz produktov proteoliza geksonov adenovirusov primatov.

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.

[Electrophoretic purification of biologically active structural proteins of the simian adenovirus SA7 in the presence of sodium dodecyl sulfate]. / Elektroforeticheskaia ochistka biologicheski aktivnykh strukturnykh belkov adenovirusa obez'ian SA7 v prisutstvii dodetsilsul'fata natriia.

The modification of disc electrophoresis technique in polyacrylamide gel with sodium dodecylsulphate (SDS) has been elaborated for synchronous isolation of some structural proteins in biologically active form and in preparative quantities from adenoviruses. Virions of SA7 adenovirus were mildly dissociated in SDS solution at 20 degrees C and structural proteins were stained by fluorescamin. After separation the zones of proteins corresponding to the native capsomeres of hexon and protein IV as well as the zones of inner proteins V and VII have been identified as fluorescent at UV-irradiation, excised and extracted by SDS solution. After the removal of SDS by protein precipitation in acetone the preparations of hexon and IV reveal the quaternary structure of native capsomers and full spectrum of antigenic and immunogenic activities of native proteins. Preparations of inner proteins V and VII possess activity in condensing adenoviral DNA. The technique is usable for preparative purification of inner polypeptide VI SA7, as well as capsomers and inner proteins of other adenoviruses.

[Conditions of stability for the quaternary structure and antigenic activity of adenovirus hexon]. / Usloviia stabil'nosti chetvertichnoi struktury i antigennoi aktivnosti geksona adenovirusov.

The procedure of SDS-PAGE was modified by lowering the temperature of protein sample dissociation to allow the separation of denaturated adenoviral hexon chains and native hexon capsomers (trimers) in the same gel. By combining the modified SDS-PAGE with dot and blot radioimmunoassays, the range of stability of the simian adenovirus SA7 hexon quaternary structure and its antigenicity was studied against a number of physical and chemical agents known to dissociate and denaturate proteins. A perfect correlation was found between the hexon native quaternary structure (trimer) and its immunoreactivity with anti-hexon immunoglobulins. The pattern of hexon trimer stability to a wide spectrum of denaturants suggests that its subunits are held together, mainly by hydrophobic interactions, in such a way that the innersubunit contact regions make up the "hydrophobic core" of the hexon molecule.

[Effect of nuclease S1 on DNA of the highly oncogenic simian adenovirus SA7(C8)]. / Izuchenie deistviia nukleazy S1 na DNK vysokoonkogennogo adenovirusa obez'ian SA7(C8).

The effect at specific nuclease S1 on DNA and the complex viral DNA-terminal protein of the highly oncogenic simian adenovirus SA7(C8) was studied. It was shown that nuclease S1 did not digest the bound between DNA and terminal protein in the complex but residual amino acid(s) was cleaved out after digestion with pronase. The DNA obtained after nuclease S1 action could be ligated and its 5'-ends were phosphorylated by polynucleotide kinase.

[Radioimmunological analysis of simian adenovirus SA7 hexon]. / Radioimmunologicheskii analiz geksona adenovirusa obez'ian SA7.

Direct and competitive radioimmunoassay (RIA) identified several antigenic determinants in hexone of adenovirus (Ad) of lower primates SA7: a species-specific, one common for Ad SA7 and SV38, as well as 2 genus-specific determinants: one common for the studied human Ad types 1 and 6, simian Ad SA7 and SV38, cattle AD of type 3, and a new determinant common for human Ad type 6 and simian Ad SA7 and SV38. It is proposed that the above genus specific determinants be designated alpha 1 and alpha 2, respectively. Indirect evidence of the occurrence in nature of rabbit Ad was obtained as sera from some nonimmunized animals precipitate purified labeled SA7 hexone, i.e. contain antibodies to the genus-specific determinant of the hexone.

[Oncogen localization in the DNA composition of simian adenoviruses. II. The identification in SV20 virus DNA of a specific fragment possessing transforming activity]. / K voprosu o lokalizatsii onkogena v sostave DNK adnovirusov obez'ian. II. Identifikatsiia v DNK virusa SV20 spetsificheskogo fragmenta, obladaiushchego transformiruiushchei aktivnost'iu.

Simian adenovirus 20 DNA was specifically cleavered by restriction endonucleases EcoRI, BamHI, XbaI and HindIII. The transformation activity of the DNA digest was investigated. BamHI, XbaI, and HindII DNA digests were able to transform the primary rat kidney cell culture (Wistar) as well as the native SV20 DNA. The transforming activity was revealed in a specific fragment of the viral DNA, obtained after the treatment of the DNA with BamHI (fragment B), with molecular weight 5.4 x 10(6) dalton. This fragment is located in the left end of the viral genome. The lack of cell transformation by the EcoRI-hydrolysate of viral DNA may serve a proof of the extremely left position of the oncogene in the viral genome, since of EcoRI-fragment chips off a fragment with molecular weight 3 x 10(5) dalton fr om the left side of DNA molecule.

[Oligopeptide analysis of the hexons of human adenovirus types 1, 2 and 6 and simian adenovirus SA7]. / Oligopeptidnyi analiz geksonov adenovirusov cheloveka tipov 1, 2, 6 i adenovirusov obez'ian SA7.

A comparative analysis of hexons of human adenovirus (HAdV) types 1, 2, 6 and simian adenovirus type 7 (SA7) was carried out by peptide mapping. Common and unique peptides were found in hexons from virions of human adenovirus types 2 and 6 and SA7. The similarity of hexons of HAdV types 2 and 6 was shown to be more marked than HAdV and SA7 hexons. Similar data were obtained in the analysis of excessively synthesized (soluble) hexons of HAdV types 1 and 6 and SA7. A significant homology between structural and soluble hexons of HAdV-6 HAdV-6 was established, and several peptides unique for each of them were discovered. The significance of the experimental results is discussed.

[Identification of a new antigenic variant of simian adenovirus (SV30N)]. / Identifikatsiia novogo antigennogo varianta adenovirusa obez'ian (SV30N).

A new variant of simian adenovirus SV30-N is described. The variant is antigenically close to SV30 virus in the neutralization test but has some antigenic relationship to SA7 virus. The properties of SV30-N virus were retained after 5 clonings by the plaque method, after passage and cloning of the virus treated with immune sera to SV30 or SA7. The heteroduplex analysis showed DNA of the virus under study to be by 80% and 40% homologous to SV30 DNA and SA7 DNA, respectively, whereas no heteroduplex molecules between SV30 and SA7 DNA were found. By the set of polypeptides the antigenic variant SV30-N is close to SV30 but differs from SV30 and, particularly from SA7. Unlike SV30 and SA7 viruses, the SV30-N virus showed no oncogenic activity for hamsters.

[Peptide mapping of the hexon and internal protein of type 6 human and type 7 simian adenoviruses]. / Peptidnoe kartirovanie geksona i vnutrennego belka adenovirusov tipa 6 cheloveka i tipa 7 obez'ian.

Hexons (polypeptide 11) and core proteins (polypeptide V) of human adenovirus type 6 and simian adenovirus type 7 were isolated using electrophoresis in polyacrylamide gel. Tryptic peptide maps showed the similarity but not identify in the structure of proteins studied. This method seems to be the most suitable for comparison of relative viral proteins.

[Analysis of the structural proteins of human and simian adenoviruses]. / Analiz strukturnykh belkov adenovirusov obez'ian i cheloveka.

Disk electrophoresis in polyacrylamide gel was used to study the polypeptide composition of purified virions of simian adenoviruses SA7, SV20 (H), and SV 38 as well as human adenovirus type 6. The general electrophoretic pattern of separation of structural polypeptides of simian and human adenoviruses was shown to be similar; the role of a number of components in the virion was elucidated. All the simian adenoviruses under study were found to have the following characteristic sizes of polypeptide components: components V and VII (DNA-framework) 50-51 and 18-19 kilodaltons, respectively, polypeptide VI (prihexon component) 29-30 kilodaltons. Simian adenovirions differ from all human adenovirus serotypes studied in the size of component VI. Capsid components (polypeptides II, III, and IV) vary in size in individual serotypes of simian adenoviruses, their set being unique for each serotype and allowing to distinguish simian adenoviruses by their electrophoretic patterns.

[Simian adenovirus 20 genome. I. DNA mapping using restriction enconucleases BamHI, EcoRi, XbaI, HindIII]. / Izuchenie genoma adenovirusa obez'ian SV20. I. Postroenie tizicheskikh kart s pomoshch'iu restriktaz BamNI, EcoRI, KbaI, SalI HindIII.

The effect of specific restriction endonuclease on the simian adenovirus SV20 DNA was studied. It was shown that endonucleases SalI, XbaI, EcoRI, BamHI, HindIII cleaved the viral DNA into 3, 4, 5, 5, 8 specific fragments respectively. The sequence of fragments (physical map) was determined and found to be B-C-A for enzyme SalI, C-D-B-A--for enzyme Xbal, E-A-C-D-B--for enzyme EcoRI, B-E-C-A-D--for enzyme BamHI and B-E-A-C-(GH)-D-F--for enzyme HindIII. The G-C content of specific fragments was studied. The "right"-"left" orientation of the physical map of the simian adenovirus 20 DNA based on the G-C content was made in respect with the nomenclature of human adenoviruses.

[GC-pair distribution in the DNA of simian SA7 and human type 6 adenoviruses]. / Raspredelenie GTs-par v DNK adenovirusov obez'ian SA7 i cheloveka tipa 6.

The buoyant density values in cesium chloride of DNA fragments of simian adenovirus SA7 obtained with restricting endonucleases of R. EcoRI, R. BamHI, R. SalI and human adenovirus type 6 DNA fragments obtained with restricting endonucleases of R. EcoRI and R. BamHI were determined. On the basis of the buoyant density values for these fragments the content of GC pairs in them was calculated. Using the known position of the fragments in the physical map of these DNAs, the distribution of GC pairs along the examined DNA molecules was constructed.

Physicochemical properties and restriction maps of simian adenovirus type 38 DNA.

The sedimentation constant of simian virus type 38 (SV-38) DNA was estimated to be 31.6S. The intrinsic viscosity of DNA was on average 86.5 dl/g and the length of the molecule determined by electron microscopy was 10.6 micrometer. The average mol. wt., as determined by sedimentation and viscometry, was 21.5 x 10(6), which agreed well with the value derived from the length of the molecule (21.4 x 10(6)) and with the value of 21.2 x 10(6) determined by the relative electrophoretic mobility of the DNA fragments produced by restriction endonucleases EcoR1, SalI and BglII. The buoyant density of the DNA in caesium chloride and casesium sulphate was 1.7185 and 1.4295 g/ml respectively. The melting temperature of the DNA in 1 x SSC was 93.5 degrees C. The GC content calculated from p and Tm values was 59.3%. BglII cleaves SV-38 DNA at three sites producing four fragments with mol. wt.: A, 9.3 x 10(6), B, 5.6 x 10(6), C, 3.3 x 10(6); and D, 2.9 x 10(6). After treatment with EcoR1 and SalI, SV-38 DNA is cleaved into five and six fragments respectively, with mol. wt. for EcoR1 fragments: A, 8.2 x 10(6), B, 6.5 x 10(6), C, 4.0 x 10(6), D, 1.27 x 10(6); and E, 1.07 x 10(6), and for SalI fragments: A, 6.5 x 10(6); B, 5.4 x 10(6); C, 4.2 x 10(6), D, 2.8 x 10(6); E, 2.5 x 10(6) and F, 0.25 x 10(6). The sequence of fragments within the SV-38 DNA molecules for BglII was deduced to be BDCA, and for ECOR1--BCEAD.

[Physicochemical properties of the DNA of simian adenovirus type 38]. / Fiziko-khimicheskie svoistva DNK adenovirusa tipa 38 obez'ian.

The sedimentation constant, specific viscosity, and the length of DNA molecule of simian adenovirus type 38 were measured and the molecular weight was estimated. The melting temperature and buoyant densities of this virus DNA in cesium chloride and cesium sulphate density gradients were determined, and the content of GC-pairs was calculated.

Characterization of DNA-protein complexes from simian adenovirus SA7.

DNA-protein complexes prepared from purified simian adenovirus SA7 virions and from lytically infected monkey kidney cells exhibited similar properties when compared with respect to size by sucrose gradient centrifugation, to configuration by electron microscopy, and to susceptibility to a variety of treatments by electron microscopy and electrophoresis in agarose gels.

Demonstration of infectious DNA in transformed cells. III. Correlation of detection of infectious DNA-protein complexes with persistence of virus in simian adenovirus SA7-induced tumor cells.

Primary tumors induced in newborn hamsters by simian adenovirus SA7 were investigated in transfection experiments. Infectious DNA-protein complexes were readily detected in both the supernatant and pellet fractions obtained by a modified Hirt extraction procedure; DEAE-dextran was required for infectivity to become manifest. Infectivity could be abolished by exposure to DNase, but it was unaffected by SA7-specific antiserum or RNase, and only partially inactivated by trypsin treatment. SA7 tumor cells serially passaged either in tissue culture or in hamsters yielded infectious DNA complexes much less frequently and appeared to evolve into nonyielder cell lines. When large numbers of cells were lysed, intact virus could be recovered from all the primary tumors and from some of the subcultured cell lines. There was a correlation between the persistence of complete virus and the presence of infectious DNA-protein complexes. When the tumor cells carried very small amounts of intact virus, infectious DNA complexes could still be detected; when virus could no longer be detected in the tumor cells, infectious DNA complexes could no longer be found. The results suggest that a portion of the infectious DNA moieties exists as viral DNA-protein complexes in the tumor cells.