Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions.

Based on physical and chemical determinations, the mnolecular weight of the type 4 adeno-satellite virus is 5.4 X 10(6) daltons, and the virion contains 1.4 X 10(6) daltons of DNA. Denaturation and renaturation studies indicate that the viral genome is a single-stranded DNA molecule and that each virion contains either a minus or a plus strand. Upon extraction, the minus and plus strands unite to form double-stranded DNA molecules with no obvious excess of unpaired strands.

Presence of protein at the termini of intracellular adenovirus type 5 DNA.

Adenovirus type 5 contains linear double-stranded DNA with protein covalently attached to the ends of the molecules. The presence of protein at the termini of intracellular viral DNA in adenovirus type 5-infected cells was investigated at different stages during the replication process. The intracellular viral DNA was isolated from the nuclei by lysis in 4 M guanidine hydrochloride. Electrophoresis on agarose gels of HsuI restriction enzyme fragments and sucrose gradient centrifugation were used to detect protein on intracellular viral DNA. After uncoating parental DNA still contains protein attached to the termini of the viral genome. Replicating and mature progeny viral DNA can also be isolated in the form of DNA-protein complexes. These complexes exhibit the same properties as the DNA-protein complex isolated from purified virions. These results suggest that the protein at the termini of intracellular viral DNA is identical to the protein attached to the 5'-ends of the DNA extracted from virions and that it is possibly involved in the replication of viral DNA.

Crystal packing and stoichiometry of the fibre protein of adenovirus type 2.

Crystals of the fibre protein of adenovirus type 2 have been grown and studied by electron microscopy and X-ray powder diffraction. The molecular packing and density of the crystals suggest that the fibre is dimeric.

Characterization of the chymotryptic core of the adenovirus DNA-binding protein.

A fragment of the DNA-binding protein of adenovirus type 5 has been obtained by controlled chymotryptic digestion of the entire molecule. Partial sequence determination indicates that the fragment consists of amino acids 174-525. The fragment is biologically active as measured by its ability to substitute for the entire molecule in a reconstituted DNA replication system. Crystals have been obtained that show diffraction to 2 A.

Analogous amino acid sequences in myelin proteolipid and viral proteins.

Computer analysis of the intrinsic membrane protein, myelin proteolipid, shows strong sequence similarities between the putative extramembrane segments of the proteolipid protein and a number of viral proteins, several of which infect humans. These similarities are even more striking than those reported previously between viral proteins and the encephalitogenic myelin basic protein (MBP). These findings, along with other reports of molecular mimicry by viruses, suggest that immunological cross-reactions between virus-induced antibodies or T-cells and analogous antigenic determinants (epitopes) in myelin proteolipid could be involved in the pathophysiology of multiple sclerosis or post-infectious demyelinating syndromes.

Simplified restriction endonuclease method for typing and subtyping adenoviruses.

Restriction endonuclease digestion of viral DNA labelled in vivo with phosphorus-32 has been used to type and to subtype both conventional and enteric adenoviruses. The method is a modification of that already described for typing and subtyping herpes simplex virus and, like the latter, needs far less material than methods previously described.

Purification of adenovirus hexon by high performance liquid chromatography.

Hexon is the major structural protein of adenovirus, and has significance in studies of virus structure and function, vaccine development, and immunodiagnosis. We describe a simple, single-step, anion-exchange high performance liquid chromatography (HPLC) method for the high yield purification of hexon. Purity of the isolated hexon was assessed by SDS-PAGE and HPLC methods. The isolated hexon was immunologically reactive with anti-hexon monoclonal antibody in a dot-blot assay. It also retained immunogenicity, as polyclonal antisera from rabbits immunized with hexon showed the desired antigen specificity. The enhanced speed of this purification method allows for the efficient isolation of hexon from various serotypes, and thus may facilitate comparative studies of hexon immunobiology.

Neurologic complications with elevated antibody titer after acute hemorrhagic conjunctivitis.

Nine patients with polyradiculomyeloneuropathy after acute hemorrhagic conjunctivitis (AHC) infection were studied serologically. All except one patient had neutralization titers larger than or equal to 1:16 against the prototype J670/71 strain of AHC virus in at least one of their serum samples. The development of a significant fourfold rise in antibody titer against AHC virus occurred in one of the patients who developed neurologic complications five days after the onset of AHC infection.

Microbial diagnosis by nucleic acid sandwich hybridization.

Sandwich hybridization is a three-component nucleic acid hybridization method suitable for the identification of microbes. In this method, one specific DNA fragment on solid support acts as catching reagent, and the second reagent is a labeled probe. The labeling of the support is mediated by a specimen nucleic acid homologous to both reagents. Because the specimen is kept in solution, relatively crude specimens not requiring elaborate pretreatments can be tested without background problems. The utility of the method in microbial diagnosis (adenovirus, cytomegalovirus, and Chlamydia trachomatis) has been demonstrated. Increased sensitivity and nonradioactive detection methods will no doubt further extend the applicability of the sandwich hybridization method.

Spot hybridization for the detection of adenoviruses and enteroviruses.

Nucleic acid spot hybridization was used for detection of adenovirus DNA directly in clinical specimens and enterovirus RNA in infected cells. The test gave results comparable to those obtained with antigen detection assays for adenoviruses. Spot hybridization could also be used for detection of enterovirus growth in cell cultures after a single passage from clinical specimens.

Soft-laser scanning densitometry performed on a 35-mm slide illustrating SDS-PAGE separation of adenovirion polypeptides.

Virion polypeptides (35 S), methionine-labeled and purified by CsCl gradient centrifugation, were separated by SDS-gel electrophoresis. Analysis of their band pattern was performed by scanning the images of the SDS-gels shown on a 35-mm slide. The densitometric tracings revealed the presence of 17 protein bands, although only 15 of them were visible to the naked eye. The high sensitivity and resolving capacities of the soft-laser scanning densitometer enabled us to detect trace amounts of protein bands separated in SDS-gels and to obtain a resolution compatible to that of electrophoresis. Fourfold electronic amplification of the densitometric tracings, produced by a computer, generated new information regarding the pattern of the electrophoregram. The facility to amplify peaks of importance is particularly advantageous when faint or overlapping protein bands revealed on a gel are assessed.

Oncogenic and nononcogenic bovine adenoviruses and guanine-cytosine content of their DNA.

Bovine adenoviruses types 1, 2, and 3 were purified and concentrated by polyethylene glycol precipitation and CsCl density-gradient centrifugation. The efficiency of recovery of infective particles was 64 to 80%. The guanine-cytosine contents of DNA of the nononcogenic types 1 and 2 and the highly oncogenic type 3 were found to be 62, 61, and 48%, respectively--a pattern similar to that of nononcogenic and oncogenic human adenoviruses.

[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.

[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.

[DNA fragmentation of chicken adenovirus CELO by specific endonucleases R. HpaI, R. EcoRI, R. HindIII]. / Izuchenie fragmentatsii DNK ptich'ego adenovirusa CELO s pomoshch'iu spetsificheskikh endonukleaz R. HpaI, R. EcoRI, R. HindIII.

The effect of specific endonucleases on DNA chiken adenovirus CELO was studed. It was shown that endonucleases R. HpaI, R. EcoRI and R. HindIII cleaved viral DNA into 5,7 and 9 specific fragments, respectively. The sequence of fragments (physical map) was determined and found to be: D-A-E-C-B for enzyme R. HpaI; B-(EG)-C-A-D-F for enzyme R. EcoRI; H-F-A-C-G-B-D-E-I for enzyme R. HindIII.

[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.

Two new formulas for triangulation number T.

The present paper reports two new formulas for calculating triangulation number T. T = L2/l2 and T = 1.45 r2/l2, where L is the distance between pentons, l the distance between any two adjacent capsomeres, r the radius of viral nucleocapsid. The formulas have been verified and applied. It is worth noticing that the triangulation number, viral size and distance between capsomeres are fully connected by the formula r/the square root of Tl = 0.83, and the capsid parameters of all icosahedral viruses are unified in one constant, 0.83.

[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.

[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.