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.

A protein serologically and functionally related to the group C E3 14,700-kilodalton protein is found in multiple adenovirus serotypes.

A 14.7-kilodalton protein (14.7K protein) encoded by the E3 region of group C adenoviruses has been shown to protect virus-infected fibroblasts from lysis by tumor necrosis factor (TNF) (L.R. Gooding, L.W. Elmore, A.E. Tollefson, H.A. Brady, and W.S.M. Wold, Cell 53:341-346, 1988). In this study we show that adenoviruses of other groups are also protected from TNF-induced cytolysis. Representative serotypes of groups A, B, D, and E produce a protein analogous to the 14.7K protein found in human group C adenoviruses. Deletion of this protein in group C viruses permits virus infection to induce cellular susceptibility to TNF killing. As with group C adenoviruses, cells infected with wild-type adenoviruses of other serotypes are not killed by TNF and are protected from lysis induced by TNF plus cycloheximide. However, cells are susceptible to TNF-induced lysis when infected with adenovirus type 4 mutants from which the 14.7K gene has been deleted. Although all known adenovirus serotypes infect epithelial cells, adenoviruses cause several diseases with various degrees of pathogenesis. Our findings suggest that the 14.7K protein provides a function required for the in vivo cytotoxicity of many adenoviruses independent of the site of infection or degree of pathogenesis.

Comparison of adenoviral hexon polypeptides (monomers) and of native hexons (trimers) by SDS-polyacrylamide gel electrophoresis.

Purified hexons of 27 serotypes of human, simian, bovine and avian adenoviruses were analysed by SDS-PAGE. The apparent molecular weights of hexon polypeptides calculated by comparison with 5 non-hexon and 3 sequenced hexon polypeptide markers ranged from 98 kDa (for bovine adenovirus Ad bos7) to 118 kDa (for simian adenovirus Ad sim13; SV36). A stability of native hexon capsomers (trimers) in SDS at room temperature permitted us to resolve native (trimeric) hexon by SDS-PAGE and to distinguish them from denatured (monomeric) hexon polypeptides by electrophoretic mobilities. Hexon trimer bands with slow mobility in SDS-PAGE (unlike hexon monomer polypeptide bands) retained native hexon antigenicity as revealed by immunoblot analyses. Possible applications of simultaneous analyses of hexon trimers and monomers by SDS-PAGE are discussed.

Adenovirus L1 52- and 55-kilodalton proteins are required for assembly of virions.

A variant of adenovirus type 5 that contained a mutation within the L1 52- and 55-kilodalton (52/55K) protein-coding region was isolated. The mutant, termed ts369, produced L1 52/55K proteins with a two-amino-acid substitution and was temperature sensitive. Temperature-shift experiments indicated that the ts369 defect was late in the viral growth cycle. DNA replication and synthesis of late proteins occurred normally in ts369-infected cells at the nonpermissive temperature, but mature virions were not produced. Rather, capsidlike particles associated with the left-terminal region of the viral chromosome accumulated. These incomplete particles could not be chased into mature virions when the infected cells were shifted to the permissive temperature. However, previously synthesized proteins could be assembled into virions in the presence of a protein synthesis inhibitor upon shiftdown from the nonpermissive temperature, suggesting that the inactivation of the L1 52/55K proteins was reversible. These results indicate that the adenovirus L1 52/55K proteins play a role in the assembly of infectious virus particles.

Biochemical analysis of adenovirus type 5 DNA-binding protein mutants.

We previously reported the isolation and functional characterization of seven adenovirus type 5 (Ad5) DNA-binding protein (DBP) point mutants (Quinn, C. O., and Kitchingman, G. R. (1986) J. Virol. 60, 653-661). Six of the seven mutants were defective in their ability to help adeno-associated virus replicate its DNA. To determine the level at which the mutations affect this function of the DBP, we analyzed several properties of the mutant proteins. All are transported to the nucleus and are post-translationally phosphorylated to the same extent. The half-lives of the proteins, measured by pulse-chase, were nearly identical to that of the wild-type DBP. The mutant DBPs were examined for their ability to bind to single-stranded DNA (ssDNA). Mutations in amino acids 322, 323, and 470 lowered the affinity of the DBP for ssDNA, while a mutation in amino acid 181 had no affect. Combinations of mutations in amino acid 470 with either 322 or 323 did not further lower the affinity of the protein for ssDNA. These data indicate that the functional defect for adeno-associated virus helper activity of the six mutants is due mainly, if not totally, to their reduced affinity for single-stranded DNA. These experiments have thus identified a functional domain of the adenovirus type 5 DBP potentially involved in DNA-protein interactions. Comparisons with temperature-sensitive DBP mutants indicate that the conserved region mutants are functionally distinct and represent a new class of DBP mutants.

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.

Identification of the phosphorylation sites in early region 1A proteins of adenovirus type 5 by amino acid sequencing of peptide fragments.

We have mapped the positions of three of the phosphorylation sites on the 289 and 243 residue (289R and 243R) early region 1A (E1A) proteins of human adenovirus type 5 (Ad5). These proteins, which play roles in both transcriptional control and oncogenic transformation, have identical sequences except for the presence in 289R of 46 additional internal amino acids. Phosphorylation was detected exclusively at serine residues. E1A proteins purified from [35S]methionine- or [32P]orthophosphate-labeled Ad5-infected cells were digested with trypsin, and two phosphopeptides were isolated by reverse-phase chromatography and subjected to automated Edman degradation. The major species was shown to contain a single phosphorylation site at Ser-219. The second phosphopeptide was shown to contain at least one phosphorylation site at Ser-231. A third phosphorylated tryptic peptide could not be eluted from the column but was isolated using an E1A-specific rat monoclonal antibody. Following subcleavage by Staphylococcus aureus V-8 protease, this peptide was shown to contain at least one phosphorylation site at Ser-89. The present data indicate that both the 289R and 243R E1A proteins are phosphorylated at the same sites, at least one in the amino terminal half of the molecule, and at least two toward the carboxyl terminus.

Use of Ar+ plasma etching to localize structural proteins in viruses: studies with adenovirus 2.

The experiments described here were undertaken to test the idea that low energy Ar+ plasma etching could be employed as the basis of a method to order viral structural polypeptides according to their physical proximity to the virus surface. Since low energy (500 eV) Ar+ ions do not penetrate deeply into virus surfaces, one expects that the outermost proteins will be damaged before internal ones when intact virions are irradiated. To test this expectation, we exposed adenovirus 2 to a 500-eV Ar+ plasma and then employed sodium dodecyl sulfate-polyacryl-amide gel electrophoresis to assess the extent of damage to the major structural polypeptides. Gel analyses showed that the proteins exposed on the virus surface (proteins II, III, and IV) were degraded rapidly during the first 10 s of irradiation while protein VII, the major core polypeptide, was almost completely protected. Proteins located between the capsid and the core, such as proteins IIIa and VI, were degraded at intermediate rates. Quantitative measurements demonstrated that the observed decay rate differences were not due simply to differences in protein target size; distance to the virion surface made an important contribution. The plasma etching technique, therefore, appears to have considerable potential for the structural analysis of viruses and other macromolecular assemblies where the proximity of individual proteins to the particle surface is unknown.

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.

The gene-specific initiation factor USF (upstream stimulatory factor) bound at the adenovirus type 2 major late promoter: mass and three-dimensional structure.

The gene-specific transcription initiation factor USF (upstream stimulatory factor) binds at a palindromic sequence that extends from -52 to -63 relative to the start site of the adenovirus type 2 major late promoter; USF enhances in vitro transcription 10- to 20-fold. By analysis of digital micrographs from the Brookhaven scanning transmission electron microscope, we have identified a sample of 29 proteins (mass, 55 +/- 5 kDa) specifically bound at the palindrome. The individual protein digital images show extensive homology, which permits modeling a three-dimensional structure at a relatively low resolution, which is nonetheless significant for the study of protein-protein interactions in initiation. Non-sequence-specific competitor DNA at high mass excess can be used in reactions for microscopy, enabling characterization of specific binding for proteins present at 1% of total protein or less.

Purification of adenovirus hexon protein by high-performance liquid chromatography.

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

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.

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.

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates.

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.

[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.

Mapping of functional domains in adenovirus E1A proteins.

We have modified the E1A gene of human subgroup C adenovirus by introducing deletions in its coding sequence. Various truncated E1A proteins were expressed in Escherichia coli, purified, and microinjected via glass capillaries into Vero cells. We monitored their movement from the cell cytoplasm to the nucleus and their ability to induce expression of H5dl312, an adenovirus E1A deletion mutant. Our results show that the carboxyl terminus of E1A contains sequences essential for rapid and efficient nuclear localization. Essential information for efficient H5dl312 complementation is contained in an internal region, comprising sequences of both exons of the E1A gene. A first exon-encoded region, however, is sufficient to induce low levels of adenovirus gene expression. Information for nuclear localization and for H5dl312 complementation are therefore encoded by distinct domains of the E1A gene. In addition, we determined that the human c-myc product was unable to complement H5dl312.

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.

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.

[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.