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PubMed1987

Immunological analysis of 140-kDa adenovirus-encoded DNA polymerase in adenovirus type 2-infected HeLa cells using antibodies raised against the protein expressed in Escherichia coli.

The E2B region of adenovirus genome contains a long open reading frame (ORF) extending from 24 to 14.2 map units which encodes most of the 140-kDa DNA polymerase. It was cloned at the polylinker region of pUC18 vector with Escherichia coli JM109 as the host. A clone was serendipitously isolated that expressed in E. coli a protein of approximately 120 kDa in size at high levels. DNA sequence analysis of this clone showed the presence of an in-frame fusion of a region, encoding 13 amino acids located upstream, to the first ATG of the ORF. Polyclonal antibodies raised against this protein purified from E. coli were used for immunological analysis. The antibodies were able to detect a 140- and a 66-kDa polypeptide from the adenovirus type 2-infected HeLa cells on Western blots. In addition, the antibodies showed evidence of cross-reactivity with partially purified DNA polymerase alpha from uninfected HeLa cells. The subcellular localization of the viral polymerase in the infected HeLa cells by using indirect immunofluorescence showed that the viral protein is associated with globular structures in the nucleus. The replicating viral DNA and the polymerase were colocalized in these globular sites. Furthermore, HeLa cells infected with Ad5ts149, a temperature-sensitive mutant defective in DNA replication, showed the presence of these globular sites only at the permissive temperature, suggesting that these sites are probably involved in viral DNA replication.

Adenoviruses, Human↗
PubMed1987

E1B functions of type C adenoviruses play a role in the complementation of blocked adenovirus type 12 DNA replication and late gene transcription in hamster cells.

Adenovirus type 12 (Ad12) DNA cannot replicate in hamster cells and the late Ad12 genes cannot be expressed. It has been demonstrated previously that these defects can be at least partly overcome by coinfection of hamster cells with Ad12 and wild-type adenovirus type 2 (Ad2) or type 5 (Ad5) or by superinfection of Ad2- or Ad5-transformed cells with Ad12. These transformed cell lines carry in an integrated form and constitutively express the E1 region of Ad2 or Ad5. The compensation in Ad12 DNA replication and late gene transcription does not, however, lead to the assembly of intact Ad12 virions. In the present study, it has been demonstrated that the complementing functions in the Ad5 genome, which can effect Ad12 DNA replication and late transcription in hamster cells, reside predominantly but not exclusively in the E1B region. A supporting role in the E1A region is likely. These conclusions have been adduced from the results of double infection experiments using Ad12 and deletion mutants of Ad5. Inside the E1B region of Ad5 DNA, the complementing functions have not yet been precisely located. Although late Ad12 messenger RNAs are synthesized in Ad12 and Ad5-coinfected hamster cells, most of the late structural Ad12 proteins are not made or are made in minimal amounts, and consequently virions are not assembled. It is necessary to investigate whether hamster cells also exhibit a translational block vis à vis the expression of late Ad12-specific mRNAs. The data presented here also demonstrate that Ad12 functions can effectively complement E1A or to a lesser extent E1B deletions in the Ad5 genome in hamster cells. Upon coinfection with Ad12 and deletion mutants of Ad5 in either the E1A or the E1B region, Ad5 DNA and late proteins are synthesized, although Ad5 E1A or E1B functions cannot complement the deficient late Ad12 protein synthesis in hamster cells.

Adenoviruses, Human↗
PubMed1987

Nucleotide sequence of the adenovirus type 40 inverted terminal repeat: close relation to that of adenovirus type 5.

Human adenovirus type 40 (Ad40) is a pathogen that causes acute infantile gastroenteritis. Ad40 has the distinct characteristic of being difficult to propagate in conventional cultured human cells. The nucleotide sequence of the inverted terminal repeat (ITR) of Ad40, which includes the origin of adenoviral DNA replication, was determined using recombinant plasmid DNA. By using our newly developed program to express the ITR homologies simply, we found that the ITR of Ad40, which is 163 nucleotides long, was related most closely to that of adenovirus type 5, which replicates efficiently.

Adenoviruses, Human↗
PubMed1990

Adenovirus type 2 precursor proteins are cleaved by proteinases of other adenoviruses.

A virus-encoded endoproteinase is responsible for the cleavage of adenovirus type 2 (Ad2) precursor proteins. By analyzing the genetic maps of previously characterized interserotypic recombinants between Ad2 and Ad5 and by means of an in vitro proteinase assay using Ad1, 2, 3, 4, 5, 6, 7, 9 as the source of enzyme, it was demonstrated that (i) the enzyme was present in these serotypes and by extension it is likely to be present in all adenoviruses, and (ii) that proteolytic cleavage can take place efficiently even between widely different serotypes. These attributes suggest a high degree of sequence conservation of the enzyme and substrate cleavage sites, thus designating proteolytic processing as an excellent target for chemotherapy.

Adenoviridae↗
PubMed1992

The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2.

We report the sequence of 7558 nucleotides of the adenovirus type 5 genome. With this sequence and previously published data, the complete sequence of this genome is now available and can be compared with the already known sequence of the adenovirus type 2 genome. These two serotypes belong to the same subgroup and sequence comparison shows 94.7% homology between the two genomes. The differences are not at all randomly distributed. Transitions between C and T and between A and G account in total for 58.3% of the differences and even for 68.6% for the genome devoid of the fiber and the hexon genes (instead of 33% expected for an equal probability of changes). In the fiber gene the transitions account for 47% of the differences. The detailed analysis of the nucleotide substitution between the two genomes suggests that the Ad2 genome could derive from that of Ad5 one, with the exception of the fiber gene which is likely to be present in Ad2 genome as a result of genetic recombination. The homology between the amino acids sequences of the structural proteins varies from 100% (proteins pVII and IX) to only 69.2% for the fiber.

Adenoviruses, Human↗
PubMed1983

Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element.

A plasmid containing the adenovirus E2 gene, a gene normally requiring E1A-mediated induction during viral infection, is expressed very poorly upon transfection into mouse L cells. If the same plasmid is transfected into 293 cells, which constitutively express the adenovirus E1A gene, or into L cells together with a plasmid containing the E1A gene, the E2 gene is expressed at higher levels. Cotransfection of the E2 plasmid with a plasmid containing the pseudorabies virus (a herpesvirus) immediate early gene results in an even higher increase in the level of E2 expression. In addition, efficient E2 expression in the absence of trans induction was obtained by inserting E1A upstream promoter sequences at the 5' or 3' end of the E2 gene, indicating that these E1A sequences possess enhancer properties. Thus the efficient expression of the E2 gene can be obtained either by a structural change in the gene itself or by a trans-acting induction.

Adenoviruses, Human↗
PubMed1990

Adenovirus transformed monkey cell lines permissive to enteric adenovirus type 40.

Plasmids containing the E1 regions of adenovirus serotypes 3 and 5 were transfected into primary Rhesus monkey kidney cells. The presence of viral DNA sequences was detected in transformed cell lines. All these cell lines expressed the E1A proteins. In addition, Ad5 transformed cells, have the E1B 21 kDa protein located in the nuclear membrane. These cell lines were permissive to the enteric adenovirus serotype 40 but not to serotype 41.

Adenoviridae↗
PubMed1984

Efficient coupled transcription and mRNA splicing in vitro using plasmids derived from early region 3 of adenovirus 2 and a nondefective adenovirus-simian virus 40 hybrid.

Accurate and highly efficient (80%) splicing of a single mRNA precursor to processed products was achieved using HeLa cell extracts to synthesize and process RNA in vitro from recombinant plasmids containing specific DNA segments from adenovirus 2 (Ad2) and the nondefective adenovirus-simian virus 40 (Ad+2ND1) hybrid. One plasmid, pRID, contains a segment of Ad2 DNA spanning chromosome map coordinates 75.9-83.4. The other plasmid, pRW9, contains the analogous viral region from Ad+2ND1. RNA synthesis from pRID in vitro occurs for more than 60 min and is directed by RNA polymerase II. RNA products consistent in size with the expected precursor and the two processed mRNAs are made. RNA blot hybridization analyses showed that these products are complementary to the Ad2 insert in the plasmid and that the appropriate intervening sequence was absent from the smallest processed mRNA. Comparison of the splice patterns of RNA made in vitro to those of RNAs taken from infected cells using the nuclease S1 technique demonstrated the accuracy of intron removal.

Adenoviruses, Human↗
PubMed1991

Induction of c-fos mRNA and AP-1 DNA-binding activity by cAMP in cooperation with either the adenovirus 243- or the adenovirus 289-amino acid E1A protein.

Products of the adenovirus E1A gene can act synergistically with cAMP to activate transcription of several viral early genes and the cellular genes c-fos and jun-B. Transcription factor AP-1-binding activity is also induced by the combined action of E1A and cAMP. Mouse S49 cells were infected with adenovirus variants expressing either the 243- or 289-amino acid E1A protein and treated with the cAMP analog dibutyryl-cAMP. Significant E1A-dependent induction of c-fos mRNA and AP-1-binding activity was observed in cells expressing either E1A protein. These effects absolutely required the presence of cAMP. In contrast, the 243-amino acid protein was a poor activator of the viral early genes E2 and E4 compared with the 289-amino acid protein. These data suggest that the 243- and 289-amino acid E1A proteins both interact functionally with the cAMP signaling system to activate transcription of a cellular gene and AP-1-binding activity. The mechanism involved in this process is probably different from the mechanism of transcriptional activation of viral genes.

Adenoviridae↗
PubMed1964

IMMUNOFLUORESCENT STUDIES OF ADENOVIRUS 12 TUMORS AND OF CELLS TRANSFORMED OR INFECTED BY ADENOVIRUSES.

Complement-fixing (CF) antibody-positive sera from hamsters bearing adenovirus type 12 (Ad. 12)-induced tumors revealed specific immunofluorescent stainable antigens in essentially all Ad. 12 hamster tumor cells. The antigens were primarily in the form of cytoplasmic flecks; less frequent staining was seen as nuclear flecks or homogeneous staining of nucleus and cytoplasm of a small proportion of cells. Tumor cells did not stain with rabbit antisera to crude Ad. 12 virus or A and C antigens. The hamster serum also stained cytoplasmic flecks in an Ad. 12-induced BALB/c mouse tumor and Ad. 12-"transformed" hamster embryo tissue culture cells. The hamster serum also stained fleck-shaped antigens in hamster and human cell cultures inoculated with homologous and heterologous adenovirus types, although the hamster cells did not react with the rabbit Ad. 12 antiserum. Attempts to identify the fluorescent-stainable fleck-shaped antigens indicated that they are not previously recognized viral antigens and that the cytoplasmic antigens formed in hamster cell cultures inoculated with Ad. 12 are different from those in tumors and in acutely infected human cell cultures.

Adenoviridae↗
PubMed2000

Efficiency of adenovirus-mediated gene transfer to oropharyngeal epithelial cells correlates with cellular differentiation and human coxsackie and adenovirus receptor expression.

Adenovirus-mediated gene transfer is a novel treatment strategy for head and neck squamous cell carcinoma (HNSCC) that may improve the unacceptable morbidity and mortality associated with conventional treatment. Efficient adenoviral (AdV) infection largely depends on cellular expression of the human coxsackie and adenovirus receptor (hCAR); however, the relatively recent identification of this receptor precludes a comprehensive description of its tissue distribution. We have created tissue culture model systems that approximate the differentiation and three-dimensional structure of stratified squamous epithelium characteristic of head and neck mucosa. Using these systems, we have found that expression of hCAR in native and modeled normal oropharyngeal epithelium decreased as cells differentiated with the most superficial and differentiated cells expressing no detectable hCAR. In contrast, modeled stratified HNSCC cells, which did not differentiate morphologically and did not express cytokeratin markers of differentiation, had equivalent expression of hCAR in superficial and basal layers. The expression of hCAR in our models correlated not only with the undifferentiated state, but also with efficiency of AdV infection. Despite expression of hCAR in underlying basal and suprabasal cells, topical application of AdV to normal modeled epithelium resulted in inefficient transduction of the most superficial cell layer without any infection of underlying cells. These data suggest that in normal epithelium the overlying squamous cells act as a barrier preventing infection of underlying cells that would otherwise be easily infected. In modeled stratified HNSCC, transduction was much more efficient and occurred up to four cell layers deep, suggesting that unlike normal superficial epithelial cells, the superficial cells of stratified HNSCC do not act as an effective barrier to adenoviral infection. The distribution of hCAR in native tissue and the enhanced susceptibility of undifferentiated oropharyngeal epithelial cells, including undifferentiated cancer cells, to AdV infection has important implications for the development of AdV-based targeting strategies for the treatment of head and neck cancer or premalignancies.

Adenoviridae↗
PubMed1995

Trans-complementation of E1-deleted adenovirus: a new vector to reduce the possibility of codissemination of wild-type and recombinant adenoviruses.

Treatment of cystic fibrosis by gene therapy will require the development of vectors capable of efficient and safe transfer of a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. To achieve this goal, replication-deficient (E1-) adenoviruses (Ad) are promising vectors. We have previously demonstrated efficient CFTR gene delivery to the airways of cotton rats and rhesus monkeys using a replication-deficient adenovirus, Ad-CFTR. Here, we have investigated an important safety issue, the interaction between the vector and wild-type virus which can provide the missing E1 function in trans. We show that Ad5 can mobilize the defective Ad-CFTR genome in vitro and in cotton rats. However, the extent of the complementation in vivo by wild-type virus is limited because no additional spreading or shedding of Ad-CFTR to trachea, lungs, and stools is elicited. To attenuate Ad-CFTR further, a mutation was introduced in the cis-acting regulatory sequences that control the encapsidation of the viral genome. We demonstrate that when cells are coinfected with wild-type virus and the new attenuated vector, the viral DNA containing the natural encapsidation sequences is preferentially packaged, leading to a rapid dilution of the recombinant virus.

Adenoviridae↗
PubMed1989

Mechanism of inhibition of adenovirus DNA replication by the acyclic nucleoside triphosphate analogue (S)-HPMPApp: influence of the adenovirus DNA binding protein.

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [S]-HPMPA) is a potent and selective inhibitor of adenovirus (Ad) replication in cell culture. We studied the mechanism of inhibition using a reconstituted in vitro DNA replication system. The diphosphoryl derivative (S)-HPMPApp, but not (S)-HPMPA, inhibited the DNA replication of origin containing fragments strongly. The inhibitory effect was exerted at the level of elongation, while initiation was resistant to the drug. Remarkably, the elongation of short strands was only slightly impaired, while inhibition was maximal upon synthesis of long DNA fragments. (S)-HPMPApp appeared to be competitive with dATP, suggesting that the Ad DNA polymerase is the prime target for the drug. We purified the Ad DNA polymerase in complex to the precursor terminal protein to homogeneity from cells infected with overproducing recombinant vaccinia viruses. Employing gapped DNA or poly(dT).oligo(dA) templates, only a weak inhibition was observed. However, inhibition was strongly enhanced in the presence of the adenovirus DNA binding protein (DBP). We interpret this to mean that the increased processivity of the polymerization reaction in the presence of DBP leads to increased drug sensitivity.

Adenine↗
PubMed2004

Adenovirus and respiratory syncytial virus-adenovirus mixed acute lower respiratory infections in Chilean infants.

BACKGROUND: In Chile respiratory syncytial virus (RSV) and adenovirus (AD) are the principal viruses detected in acute lower respiratory infections (ALRI) in infants. An overview of AD pneumonia in Chile to detect annual trends and to compare the severity of single AD or mixed RSV-AD infections is presented. METHODS: Surveillance in 4927 infants hospitalized for ALRI has been performed from 1989 to 2001 using immunofluorescence assay (IFA) and viral isolation. Clinical features in 117 infants with single genotyped AD and 81 infants with mixed RSV-AD infections were analyzed. RESULTS: Adenovirus cases declined from 20% annually in the early 1990s to approximately 5% in the 2000 decade. Genotype 7h showed increasing prevalence in hospitalized cases. The mean annual burden of hospitalizations caused by AD in Santiago was estimated to be 0.6%. No difference was observed in duration of fever, oxygen requirement and hospital stay between groups. Lung consolidation was more frequent in AD cases than mixed cases (P < 0.01); interstitial pattern and hyperinflation prevailed in the mixed cases (P < 0.01). No child died. AD diagnosis was confirmed on admission by IFA in 17% of cases of RSV-AD and in 43% of cases of single AD ALRI. AD cases diagnosed early by IFA had worse clinical outcome than those diagnosed later by virus isolation (P < 0.05). CONCLUSIONS: AD cases declined since 1989. Mixed RSV-AD infections were not more severe than single AD etiology. AD cases admitted with positive IFA had worse prognoses than AD infections diagnosed later by virus isolation.

Adenovirus Infections, Human↗
PubMed1982

Adenovirus-cell interactions early after infection: in vitro characteristics and tumourigenicity of adenovirus type 2-transformed rat liver epithelial cells.

Cloned rat liver epithelial cells (clone C3) were semi-permissive for adenovirus type 2 (Ad-2) and non-permissive for adenovirus type 12 (Ad-12). Ad-2-infected C3 cells were shown to produce hexon and fibre protein, but at an m.o.i. of 20 a maximum virus yield of only 2.4 p.f.u. per cell was obtained. Forty-eight h after infection with Ad-12 'early' virus proteins (major species 8K and 60K), but no 'late' proteins (virus structural proteins) could be identified. Of six Ad-2-transformed epithelial lines isolated from clone C3 only one was tumourigenic in syngeneic rats, whereas all six transformants produced tumours in athymic nude mice. There was a remarkable variation in the morphology of the Ad-2-transformed liver cells, ranging from an epithelial morphology similar to C3 cells to cells with a distinct lymphoid morphology. The in vitro and in vivo behaviour of the Ad-2-transformed clone C3 cells reported in this communication, taken together with our previous report on the characteristics of Ad-12-transformed C3 cells, clearly show that the differences observed between Ad-2- and Ad-12-transformed rat embryo cells were also observed in our studies using cloned rat liver epithelial cultures. Our findings clearly rule out the hypothesis that the heterogeneity of Ad-2-transformation events is the result of the transformation in vitro of different types of target cell.

Adenoviruses, Human↗
PubMed1990

Molecular cloning and physical mapping of the DNA of bovine adenovirus serotype 4; study of the DNA homology among bovine, human and porcine adenoviruses.

The DNA of bovine adenovirus (BAV) serotype 4, a member of the subgroup 2 BAVs, has been cloned and mapped with 11 restriction enzymes. Southern blot hybridizations probed by a clone containing about 50% of the BAV-4 genome revealed a very strong and extended DNA sequence homology amongst the members of subgroup 2, but no homology was detectable to the subgroup 1 bovine, or any of those human (HAV) and porcine adenovirus serotypes examined. These findings were strengthened by reciprocal hybridizations. When using the cloned hexon gene region of BAV-3 (subgroup 1) or the total genome of HAV-2 as probes, again no homology could be shown to the bovine subgroup 2 serotypes. The extent of DNA homology detectable between the members of bovine subgroup 1, the porcine and the human serotypes was variable, but in general less expressed than that observed within the bovine subgroup 2.

Adenoviridae↗
PubMed1981

Grouping of adenoviruses and identification of restriction endonuclease cleavage patterns of adenovirus DNAs using infected cell DNA: simple and practical methods.

Simple and practical methods for grouping of adenoviruses and for identification of restriction endonuclease cleavage patterns of viral DNA were established by using infected cell DNA. DNA homology groupings of adenoviruses could be examined by spot hybridization, and restriction endonuclease cleavage patterns of viral DNAs could be obtained by Southern blot hybridization, by using infected cell DNA. The method was very sensitive and allowed the identification of the cleavage pattern of viral DNA of the inoculum by means of cell DNA extracted from infected cells with undetectable cytopathic effect (CPE). In ethidium bromide-stained gels without Southern blot hybridization, the restriction endonuclease cleavage pattern of viral DNA could be detected precisely in spite of background staining due to cellular DNA. The preparation of infected cell DNA used in these procedures was technically much easier than that of viral DNA. These methods require only a small number of infected cells and allow many isolates to be investigated with ease.

Adenoviruses, Human↗