Modulation of E-cadherin localization in cells expressing wild-type E1A 12S or hypertransforming mutants.
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The adenovirus E1A 12S gene can immortalize primary epithelial cells such that they retain expression of epithelial cell characteristics. E1A 12S can also cooperate with an activated ras oncogene to cause tumorigenic transformation of primary cells. Specific substitution and deletion mutants of E1A 12S cooperate more efficiently with ras to produce foci with a hypertransformed phenotype, wherein the cells are less differentiated and exhibit invasive properties. Loss of epithelial differentiation in carcinomas is often a consequence of reduced intercellular adhesion involving loss of a functional cell-cell adhesion molecule, E-cadherin. Therefore, expression of E-cadherin was analyzed in mock-infected primary epithelial cells, cells expressing E1A 12S protein, and in hypertransformed cells. Primary epithelial cells express E-cadherin and correctly localize it to the cell-cell junctions, as detected by immunofluorescence. The level of E-cadherin expression decreases with time in culture. Primary cells expressing the E1A 12S protein maintain the expression and correct localization of E-cadherin. This effect of E1A 12S on E-cadherin expression is not at the transcriptional level. Immortalized cells expressing the 12S protein and primary epithelial cells transformed with the E1A 12S gene and ras continue to express E-cadherin at the cell-cell junctions. In contrast to the 12S-immortalized and wild-type transformed cells, hypertransformed cells are defective for localization of E-cadherin and exhibit altered subcellular distribution of E-cadherin, as detected by immunoprecipitation with an anti-E-cadherin antibody. Furthermore, the aberrant localization exhibited by the hypertransformed cells can be overcome by superinfection of the hypertransformed cells with a virus expressing the E1A 12S cDNA or treatment with retinoic acid. Thus, the E1A 12S protein appears to act as a differentiation factor to maintain the differentiated characteristics of epithelial cells. (+info)
Pathology and immunogenicity in the cotton rat (Sigmodon hispidus) model after infection with a bovine adenovirus type 3 recombinant virus expressing the firefly luciferase gene.
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The histopathology of adenovirus pneumonia in cotton rats (Sigmodon hispidus) due to bovine adenovirus type 3-luciferase recombinant virus (BAd3-Luc), which has a 0.7 kb deletion from the early region 3 (E3) replaced with the firefly luciferase gene, was compared with that produced by the parental wild-type (wt) bovine adenovirus type 3 (BAd3). After intranasal inoculation of cotton rats with 3 x 10(7) p.f.u. of BAd3-Luc, the infectious virus titres in the lungs at various times post-infection were similar to those of animals infected with the parental virus. Quantitative analysis of histopathological changes and immunohistochemical staining showed that the character and severity of the lesions were indistinguishable in the two infections. Luciferase activity was detected in the lungs of BAd3-Luc-inoculated animals until 4 days post-infection (p.i.). Antibodies to both BAd3 and luciferase were detected in sera collected from BAd3-Luc-infected animals until at least 6 weeks p.i. These results show that Bad3-Luc produces pulmonary lesions in cotton rats similar to those of wt BAd3 and suggest that BAd3-based vectors may be suitable for the development of live recombinant virus vaccines. (+info)
LMP-associated proteolytic activities and TAP-dependent peptide transport for class 1 MHC molecules are suppressed in cell lines transformed by the highly oncogenic adenovirus 12.
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Expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the in vivo oncogenicity of this virus. In primary embryonal fibroblasts (H-2b) that express transgenic swine class I antigen (PD1), Ad12-mediated transformation results in inhibition in transport of newly synthesized class I molecules, as well as significant reduction in transporter associated with antigen presentation (TAP) gene expression. In this report we show that reexpression of TAP molecules either by stable transfection of mouse TAP genes or by infection with recombinant vaccinia viruses expressing human TAP genes, only partially reconstitutes the expression and transport of the class I molecules. Further analysis of Ad12-transformed cells revealed that the expression of both LMP2 and LMP7, but not of other proteasome complex components, was downregulated, resulting in altered proteolytic activities of the 20S proteasomes. Reconstitution of both TAP and LMP expression resulted in complete restoration of PD1 cell surface expression and enhanced expression of the endogenous H-2D(b) molecules encoded by recombinant vaccinia viruses, in reconstituted Ad12-transformed cells, efficient transport of H-2 class I molecules could only be achieved by treatment of the cells with gamma-interferon. These data suggest that an additional factor(s) that is interferon-regulated plays a role in the biosynthetic pathway of the class I complex, and that its function is deficient in this cell system. Thus, Ad12 viral transformation appears to suppress the expression of multiple genes that are important for antigen processing and presentation, which allows such transformed cells to escape immune surveillance. This coordinate downregulation of immune response genes must likely occur through their use of common regulatory elements. (+info)
Unique genome arrangement of an ovine adenovirus: identification of new proteins and proteinase cleavage sites.
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The completed sequence and genome organization of OAV287, a serologically distinct ovine adenovirus, is described. The genome of 29,544 bp has inverted terminal repeats that are only 46 bp in length. Many OAV genes are identified by their homology with other adenovirus (Ad) sequences but three groups of reading frames show little homology. One group at the left-hand end of the genome probably represents the E1A/E1B regions. Two others, on the complementary strand at the right-hand end of the genome, are tentatively proposed as the E4 and E3 regions. They are separated by approximately 1 kb of A/T-rich sequence of unknown function with E3 being adjacent to the terminus. Structural proteins V and IX of human Ads are absent from the OAV genome but a new, processed, 28-kDa virion polypeptide is encoded on the strand complementary to the proposed E1A region. The coding sequences for two other structural proteins are unidentified. The OAV penton protein lacks the region containing an Arg/Gly/Asp sequence that, in human adenoviruses, is thought to interact with cellular integrins to facilitate virus entry. Analysis of proteins and peptides in purified OAV identified several cleavage sites utilized by the Ad proteinase. Some of these were previously identified in human Ad proteins, but new sites, some of which did not conform to the known specificity of the human Ad proteinase, were also identified. The data emphasize that this ovine virus differs significantly from other known human and animal adenoviruses. (+info)
Construction and transfection of ovine adenovirus genomic clones to rescue modified viruses.
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The genome of ovine adenovirus OAV287 has an arrangement which is unique among known adenoviruses. To facilitate further experimentation on the structure and function of this genome, plasmids containing a complete clone of the genome were constructed. The cloned viral genome was released from plasmids by restriction enzyme digestion as an intact linear molecule with authentic 5' termini. Transfection of the linear DNA into cells which supported replication produced infectious virus. Mutation of a unique SalI site at the right-hand end of the genome disrupted reading frames of unknown function without affecting virus rescue, identifying this region as nonessential for replication in vitro. A 20-bp oligonucleotide was also inserted into the short intergenic region between the pVIII and the fiber sequences, identifying a second site for gene insertion. These studies will facilitate the development of OAV as a gene transfer vector. (+info)
Conservation of DNA sequence in the predicted major late promoter regions of selected mastadenoviruses.
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The major late promoter (MLP) of the subgroup C human adenoviruses is a preeminent model for the study of the mechanisms of basal and activated transcription, both in vivo and in vitro. However, while the structure and function of the human virus MLP has been the subject of extensive investigation, the conservation of the various promoter elements among the adenoviruses from different species has not been examined. Conservation of specific elements would strongly suggest the importance and universality of their function. To address this issue, sequences were obtained from cloned DNAs of several representative Mastadenoviridae, mouse adenovirus type 1 (MAV-1), Tupaia adenovirus type 1 (TAV-1), and two bovine adenoviruses of two distinct subgroups, BAV-3 and BAV-7. The results of the sequencing studies showed that the TATA box and an upstream inverted CAAT box are conserved in all species and that the binding site for transcription factor USF is present in all except MAV-1, in which a sequence similar to an Sp1-binding site is present at a similar position. The initiator element (INR) sequence is not well conserved, and only one or other of the two downstream activating elements, DE1 and DE2, is predicted to be present in the nonprimate virus MLP regions. Ribonuclease protection assays on RNA isolated from MAV-1-infected cells late in infection indicated that the predicted MLP is functional, and transcription initiation and splice donor sites were identified. The human virus MLP is embedded in the essential DNA polymerase sequence on the opposite DNA strand. The primary amino acid sequences of the C-terminal regions of the predicted DNA polymerases show strong conservation of sequence motifs observed in replicative polymerases ranging from prokaryotes to mammals, and additional regions of strong conservation among the adenovirus polymerases. Pairwise comparisons between the newly sequenced regions of the polymerases and previously published sequences show that BAV-7 is most dissimilar to all others, while TAV-1 has a greater similarity to the primate sequences than to the others. The sequence data from both strands were also used to construct phylogenetic trees, based on BAV-7 as the outgroup. The trees constructed from the two sets of sequences are broadly similar, showing close relationships between primate viruses, but differing in the order of divergence of TAV-1 and MAV-1 branches. (+info)
Lesions in lambs experimentally infected with ovine adenovirus serotype 6 and Pasteurella haemolytica.
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Twenty-five colostrum-deprived lambs reared in isolation were inoculated with a US variant of ovine adenovirus serotype 6 (OAV-6) strain RTS-151, Pasteurella haemolytica, or a combination of the 2 agents. Although severe pulmonary lesions were caused by each agent, the lesions were more severe and lasted longer with the combined infection. Lesions induced by OAV-6 alone developed 6-9 days after inoculation and lasted for 15 days, the length of the experiment. The lesions were characterized by suppurative inflammation at the junction of the terminal bronchioles and alveoli. Air spaces were filled with neutrophils and sloughed epithelial cells, which often contained large intranuclear inclusions. Lesions induced by P. haemolytica alone developed within 1 day and persisted for no more than 10 days and were characterized by severe pulmonary edema with variable amounts of fibrin. Lesions induced by the combined infection had aspects of each infection alone and resulted in severe disease in 4 of 8 lambs that were permitted to live more than 1 day after inoculation with bacteria. Early pulmonary lesions included edema, limited fibrin deposition, and slight purulent bronchiolitis and alveolitis. Later lesions included necrosis and more fibrin. For lambs inoculated with both pathogens, resolution was incomplete 15 days after inoculation of virus (10 days after inoculation of P. haemolytica). The results presented here corroborate previous findings indicating that the RTS-151 variant of OAV-6 is common in lambs and acts in concert with P. haemolytica to cause severe and often fatal pneumonia. (+info)
Interaction of mouse adenovirus type 1 early region 1A protein with cellular proteins pRb and p107.
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We demonstrated functional associations between mouse adenovirus type 1 (MAV-1) early region 1A (E1A) protein and both the mouse retinoblastoma protein (pRb) and the mouse pRb-related protein, p107. Interactions between MAV-1 E1A and mouse pRb or mouse p107 proteins were examined in infected cell lysates using a mouse embryonic fibroblast cell line infected with wild-type and mutant MAV-1 viruses. Using a polyclonal antibody to MAV-1 E1A, exogenously added mouse pRb or mouse p107 was coimmunoprecipitated from wild-type, dIE105 (CR1 delta)-, and dIE106 (CR3 delta)-infected cell lysates. No coimmunoprecipitation was seen with cell lysates from dIE102 (CR2 delta) or pmE109, a mutant virus that produces no detectable E1A protein due to an ATG to TTG point mutation in the initiator methionine. Introduction of mouse pRb into SAOS-2 cells resulted in a flat and enlarged cell phenotype, whereas cotransfection of mouse pRb and MAV-1 E1A resulted in a significant reduction of flat cells, presumably due to E1A binding pRb. CR1 delta and CR2 delta E1A proteins were less effective at reducing the number of flat, enlarged cells induced by pRb expression than were the CR3 delta or wild-type E1A proteins. The reduced ability of these mutants to inactivate pRb relative to wild-type E1A correlated with their reduced ability to bind pRb in the in vitro coimmunoprecipitation experiments. As a measure of p107/MAV-1 E1A complex formation in MAV-1-infected cells, we used mobility shift assays to examine cell extracts for the presence of p107-containing E2F protein-DNA complexes. Mock-, dIE102-, and pmE109-infected cell extracts formed a p107-containing complex, whereas wild-type-infected cell extracts did not. Thus the formation of a p107-E2F complex in wild-type- or these mutant-infected extracts inversely correlated with the presence of E1A-p107 complexes identified in the vitro coimmunoprecipitation experiments. This is consistent with E1A-p107 complexes forming in wild-type MAV-1-infected cells. (+info)