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PubMed2000

Genome analysis of adenovirus type 7 and adenovirus type 11

Purpose: To study the epidemiology of adenovirus type 7 (Ad 7) conjunctivitis and adenovirus type 11 (Ad 11) conjunctivitis by determining genome types and subgenomic types.Materials and Methods: For Ad 7 I used twelve strains from patients with acute viral conjunctivitis and one strain from a patient with pneumonia. For Ad 11 I used seventeen strains from patients with cystitis. For Ad 7 genome typing, I used eleven DNA restriction endonucleases (REs) recognizing 6- or 7-base pair sequences and for Ad 11 genome typing, I used seven. For Ad 7 and for Ad 11 subgenome typing, I used Taq 1 and Hinf I which recognize 4- or 5-base pair sequences.Results: The thirteen Ad 7 strains all belonged to the same genome type and subgenome type. Ad 11 strains showed six genome types. Ad 11 p was the most frequent strain. Fifteen Ad 11 p strains showed three subgenome types, but none of them was the same as the prototype. Conclusions: Ad 7 seems quite stable and the Ad 7 epidemic may recur again. On the other hand Ad 11 showed several different types. Ad 11 was probably not epidemic in the first half of the 1990s.

Journal Article↗
PubMed1990

Structure of adenovirus fibre. I. Analysis of crystals of fibre from adenovirus serotypes 2 and 5 by electron microscopy and X-ray crystallography.

An analysis by electron microscopy in amorphous ice and X-ray diffraction of four types of three-dimensional crystals of adenovirus fibre is presented. Fibre from adenovirus type 2 (Ad2) crystallizes in two forms depending on whether it is native or cleaved near the N terminus at Tyr17. Fibre from Ad5 also crystallizes in two forms, both of which contained fibre cleaved at Tyr17. Analysis of the packing of the fibres in each of these crystals suggests that the overall length of the fibre may be considerably longer (about 350 to 370 A) than previously reported. Crystals of cleaved Ad2 fibre are of sufficient quality to be characterized by X-ray diffraction. They are of space group C2 and cell dimensions a = 134.4 A, b = 77.6 A, c = 539.4 A, beta = 92.7 degrees. These crystals are remarkable in that, despite being monoclinic, the ab plane forms a perfect hexagonal lattice. This is explained by a trigonal packing of the trimeric fibre heads in the crystal. A similar feature is found for one type of Ad5 crystal, although the hexagonal lattice is 12% smaller. The crystals of cleaved Ad2 show very strong meridional intensity at a Bragg spacing of 4.4 A and weaker diffuse intensity corresponding to layer-lines of spacing 26.4 A. This must reflect the quasiperiodicity of the structure of the fibre shaft, which is apparent in the primary sequence. The occurrence of these features combined with the new determination of the length of the fibre (see also the accompanying paper) require a reappraisal of the cross-beta model of the fibre shaft proposed by Green et al.

Adenoviridae↗
PubMed1976

DNA methylation in adenovirus, adenovirus-transformed cells, and host cells.

DNAs of adenovirus type 2 and type 12 contain low amounts of methylated bases (0.01 and 0.02% N6-methyl-adenine per adenine, if any, and 0.04 and 0.06% 5-methylcytosine per cytosine for type 2 and type 12, respectively), whereas the DNA of the mammalian host cells contains much more 5-methylcytosine (3.57% for human KB cells). The DNA of hamster cells transformed by adenovirus type 12 contains 3.11 and 3.14% 5-methycytosine (HA12/7 and T627 cells, respectively), whereas the DNA from untransformed hamster cells (BHK21 cells) contains 2.22% 5-methylcytosine. In the DNA of human and hamster cells, little, if any, N6-methyladenine was detected. Methylation of DNA was determined by a sensitive method based on two consecutive steps of two-dimensional thin-layer chromatography of the radioactively labeled DNA bases. By this procedure the detection limits of 5-methylcytosine and N6-methyladenine could be lowered to 0.01% per main base.

Adenoviridae↗
PubMed1976

Intracellular forms of adenovirus DNA: integrated form of adenovirus DNA appears early in productive infection.

In KB cells productively infected with adenovirus type 2, alkali-stable greater than 100S and 40-100S viral DNAs are synthesized starting 2-4 hr postinfection, i.e., before unit length (34 S) viral DNA is made. The amount of greater than 100S and 40-100S viral DNA increases when 34S viral DNA synthesis begins, and at 16-18 hr postinfection, the 40-100S viral DNA represents 5-20% of the total intracellular viral DNA. The 40-100S viral DNA is synthesized throughout infection. Part of the 40-100S DNA synthesized 5-8 hr postinfection has a density in alkaline CsCl gradients intermediate between those of viral and cellular DNAs. This finding indicates that newly synthesized viral DNA is covalently linked to cellular DNA. Viral sequences can be excised from the cellular DNA of infected cells with the EcoRI restriction endonuclease. Fragments of viral DNA are detected in polyacrylamide-agarose gels by DNA-DNA hybridization, and these fragments correspond in size to most of the known EcoRI fragments of adenovirus 2 DNA. Viral DNA sequences in size-classes between the EcoRI-A and -C fragments are also found and probably represent viral DNA linked to cellular sequences.

Adenoviridae↗
PubMed1985

The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome.

Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter. This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.

Adenoviridae↗
PubMed1998

The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures.

Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer. The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment. Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules. We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency. Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry. AdV-mediated IL-12 expression was measured using a commercial ELISA. Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta3 and alpha(v)beta5 integrins, and HLA class I. In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50. Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR. In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12. Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.

Adenoviridae↗
PubMed1998

Analysis of the hexon gene sequence of bovine adenovirus type 4 provides further support for a new adenovirus genus (Atadenovirus).

The putative hexon gene of bovine adenovirus type 4 (BAV-4), encoding 910 amino acid residues, has been identified and sequenced. A characteristic codon usage biased towards the use of AT-rich triplets was observed. Comparative analysis with other hexon sequences detected a high level of amino acid identity in the regions corresponding to the pedestals of the hexon. Substitutions, insertions and deletions were identified mainly in the variable regions forming the loops which are exposed on the outer surface of the virion. In these variable regions, BAV-4 shared similarity only with egg drop syndrome (EDS) virus and ovine adenovirus isolate 287 (OAV287). The close relationship of these viruses was also demonstrated by phylogenetic analysis of the hexon gene. In addition to the two groups of the Mastadenovirus and Aviadenovirus genera, a third cluster appeared comprising BAV-4, OAV287 and EDS virus.

Amino Acid Sequence↗
PubMed1996

Adenovirus vector technology: an efficient method for constructing recombinant adenovirus and on/off switching of gene expression.

An efficient method of constructing recombinant adenoviruses (Ad) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique SwaI site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected to 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the restriction digestion drastically reduced regeneration of the parent virus. This method may facilitate the application of recombinant Ad and should be useful for further improvement of Ad vectors. Also a recombinant adenovirus expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ad showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1 cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in future gene therapy.

Adenoviridae↗
PubMed1974

Intracellular forms of adenovirus DNA. 3. Integration of the DNA of adenovirus type 2 into host DNA in productively infected cells.

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.

Adenoviridae↗
PubMed1974

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. Transformation of hamster kidney cells by adenovirus 2 and the nondefective hybrid viruses.

Nonhybrid adenovirus 2 (Ad2) and five nondefective Ad2-simian virus 40 (SV40) hybrids (Ad2(+)ND(1)-Ad2(+)ND(5)) failed to produce tumors when injected into newborn hamsters. However, each of these agents transformed hamster kidney cells in vitro, and the transformed cell lines produced tumors when injected into suckling hamsters. Foci of cells transformed by the hybrids could not be distinguished from foci of cells transformed by nonhybrid Ad2. Histopathologically, tumors induced by the nondefective hybrid-transformed lines were of the adenovirus type with no areas of SV40-type fibrosarcoma. All of the cell lines studied contained Ad2 T antigen and Ad2 RNA. Only the B55HK(1) line contained the three early SV40 antigens, U, TSTA, and T. The ND(4)HK(1) line contained SV40 T and TSTA antigens (the latter antigen being detected only after 48 to 49 tissue culture passages) and SV40 RNA. The ND(2)HK(2) line contained SV40 RNA but no detectable SV40 antigens; the other hybrid-transformed lines contained no SV40 RNA. The transformation of hamster cells by the nondefective Ad2-SV40 hybrids seemed to be caused by the Ad2 genome and could not be attributed to the regions of the SV40 genome contained within the hybrids. Additionally, these results indicate that interactions between the genomes of the nondefective hybrid viruses and the hamster cells they transform are complex and could involve the site of integration, the size of the incorporated viral genome, the transcription or translation of the viral genome, or various combinations of these processes.

Adenoviridae↗
PubMed1975

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.

Adenoviridae↗
PubMed1975

Adenovirus gene function required for induction of nuclear acidic protein synthesis: binding of these proteins to adenovirus DNA.

Two different viral DNA-defective temperature-sensitive mutants of adenovirus 12 (H 12) were defective in their ability to induce the synthesis of various molecular weight classes of nuclear acidic proteins, both virion and nonvirion components, after lytic infection of human embryo kidney (HEK) cells at the restrictive temperature. This finding indicates that the induction of nuclear acidic protein synthesis is an adenovirus gene function(s). Treatment of infected cells with actinomycin D at an early stage of virus maturation suppressed the synthesis of an acidic virion protein (hexon), but allowed the synthesis of other classes of nuclear nonvirion acidic proteins during the subsequent late maturation period, suggesting that different mechanisms control virion and nonvirion polypeptide synthesis. The interaction of the nuclear acidic proteins isolated from H 12-infected cells with native-labeled H 12 DNA was studied using the membrane filter technique. Measurements of the ability of different DNA preparations to inhibit the H 12 DNA-acidic protein complex formation suggest that the nuclear acidic proteins bound to native H 12 or HEK cell DNA with much higher affinity than to native calf thymus DNA. Moreover, native H 12 DNA was able to bind the acidic proteins more efficiently than did denatured H 12 DNA. The acidic proteins isolated from the cytoplasm of H 12-infected cells bound approximately 100-fold less to native H 12 DNA than did the nuclear proteins. Furthermore, the H 12 DNA binding affinity of the nuclear acidic proteins from uninfected cells, or from H 12-infected and 1-beta-D-arabinofuranosylcytosine-treated cells, was somewhat lower than that of the nuclear proteins from infected (untreated) cells.

Adenoviridae↗
PubMed1976

Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of adenovirus 5.

Cytoplasmic RNA sequences produced in HeLa cells infected with the adeno-virus 5 temperature-sensitive mutants ts1, ts2, ts9, ts17, ts18, ts19, ts20, ts22, ts49, ts36, and ts125 were characterized by hybridization to DNA probes generated by strand separation of restriction endonuclease fragments of adenovirus 5 DNA. Two ""early'' mutants defective in DNA synthesis, ts125 and ts36, fail to make wild-type levels of all previously reported classes of late RNA at the nonpermissive temperature. At 40.5 degrees C, both ts125 and ts36 synthesize a wild-type complement of early cytoplasmic RNA 16 h after infection. Under these conditions, no ""late'' cytoplasmic RNA sequences were observed. Similarly, nuclear RNA present in these cells resembled early cytoplasmic RNA rather than late nuclear RNA. All the late adenovirus 5 temperature-sensitive mutants synthesized normal wild-type levels of late cytoplasmic RNA at the nonpermissive temperature, except ts2, which appears to overproduce certain cytoplasmic species.

Base Sequence↗
PubMed1988

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells.

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.

Adenoviridae↗
PubMed1994

Stimulation of the adenovirus major late promoter in vitro by transcription factor USF is enhanced by the adenovirus DNA binding protein.

Previous studies have shown that the sequence-independent adenovirus DNA binding protein (DBP) increases transcription from several promoters, notably from the adenovirus major late promoter (MLP) and the adeno-associated virus P5 promoter, both of which contain a USF/MLTF binding site. In order to study this mechanism, we have investigated the effects of DBP on the binding of USF/MLTF to MLP and on transcription from MLP by a reconstituted in vitro system. As shown by gel retardation and DNase I footprinting, upon saturation of DNA, DBP enhances the binding affinity of USF43 to the promoter three- to fourfold without changing the footprint pattern. In contrast, the binding of the TATA box binding protein to the promoter is not influenced by DBP. No protein-protein interactions between DBP and USF43 could be observed in the absence of DNA, suggesting that enhanced binding is caused by a change in DNA structure induced by the DBP-DNA complex. Employing a transcription system reconstituted with purified general transcription factors, we show that USF43 enhances basal transcription and that USF43-dependent transcription is further increased by DBP, while DBP alone does not have an effect on basal transcription. Our results suggest that transcription enhancement by DBP is based on a specific increase in the binding of a transcription factor to a promoter through subtle changes in DNA structure, similar to the mechanism by which DBP stimulates the initiation of DNA replication.

Adenoviridae↗
PubMed1998

The coxsackievirus-adenovirus receptor protein can function as a cellular attachment protein for adenovirus serotypes from subgroups A, C, D, E, and F.

Attachment of an adenovirus (Ad) to a cell is mediated by the capsid fiber protein. To date, only the cellular fiber receptor for subgroup C serotypes 2 and 5, the so-called coxsackievirus-adenovirus receptor (CAR) protein, has been identified and cloned. Previous data suggested that the fiber of the subgroup D serotype Ad9 also recognizes CAR, since Ad9 and Ad2 fiber knobs cross-blocked each other's cellular binding. Recombinant fiber knobs and 3H-labeled Ad virions from serotypes representing all six subgroups (A to F) were used to determine whether the knobs cross-blocked the binding of virions from different subgroups. With the exception of subgroup B, all subgroup representatives cross-competed, suggesting that they use CAR as a cellular fiber receptor as well. This result was confirmed by showing that CAR, produced in a soluble recombinant form (sCAR), bound to nitrocellulose-immobilized virions from the different subgroups except subgroup B. Similar results were found for blotted fiber knob proteins. The subgroup F virus Ad41 has both short and long fibers, but only the long fiber bound sCAR. The sCAR protein blocked the attachment of all virus serotypes that bound CAR. Moreover, CHO cells expressing human CAR, in contrast to untransformed CHO cells, all specifically bound the sCAR-binding serotypes. We conclude therefore that Ad serotypes from subgroups A, C, D, E, and F all use CAR as a cellular fiber receptor.

Adenoviridae↗
PubMed1999

Coxsackievirus and adenovirus receptor cytoplasmic and transmembrane domains are not essential for coxsackievirus and adenovirus infection.

Coxsackievirus and adenovirus receptor (CAR) from which the cytoplasmic domain had been deleted and glycosylphosphatidylinositol (GPI)-anchored CAR lacking both transmembrane and cytoplasmic domains were both capable of facilitating adenovirus 5-mediated gene delivery and infection by coxsackievirus B3. These results indicate that the CAR extracellular domain is sufficient to permit virus attachment and entry and that the presence of a GPI anchor does not prevent infection.

Adenoviridae↗
PubMed1999

Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase.

Production of gutted, or helper-dependent, adenovirus vectors by current methods is inefficient. Typically, a plasmid form of the gutted genome is transfected with helper viral DNA into 293 cells; the resulting lysate is serially passaged to increase the titer of gutted virions. Inefficient production of gutted virus particles after cotransfection is likely due to suboptimal association of replication factors with the abnormal origins found in these plasmid substrates. To test this hypothesis, we explored whether gutted virus production would be facilitated by transfection into cells expressing various viral replication factors. We observed that C7 cells, coexpressing adenoviral DNA polymerase and preterminal protein, converted plasmid DNA into replicating virus approximately 50 times more efficiently than did 293 cells. This property of C7 cells can be used to greatly increase the efficiency of gutted virus production after cotransfection of gutted and helper viral DNA. These cells should also be useful for generation of recombinant adenovirus from any plasmid-based precursor.

Adenoviridae↗