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Some genital human papillomavirus (HPV) types, such as 16 and 18, are highly associated with malignant cervical tumors while others, such as HPV 6, are only rarely found in these malignancies. The E7 oncoproteins of HPV 6, 16 and 18 each have a 17 amino acid region with striking homology to adenovirus E1a and SV40 LT. E1a, LT and the E7 oncoprotein of HPV16 all bind the cellular Rb protein in vitro, and for E1a and LT this region of homology contains sequences essential for interaction with Rb. We have now found that in HPV 16 E7 this region (amino acids 21-37) contains two separate biochemical activities, each of which contributes to E7-mediated transformation. Rb binding was localized to the N terminus of this region, while the C terminus was shown to serve as a substrate for casein kinase (CK) II, which phosphorylated serine-31 and serine-32. Replacement of the two serines by non-phosphorylatable amino acids led to a reduction in transforming activity and abolished phosphorylation but did not affect Rb binding. Rb binding and CK II phosphorylation were also examined for the E7 proteins of HPV 6 and HPV 18. HPV 16 and 18 E7 bound similar amounts of Rb, but HPV 6 E7 consistently bound less. Phosphorylation rates also varied, with HPV 18 E7 being 2-fold faster than HPV 16 E7, which in turn was 2-fold faster than HPV 6 E7. We conclude that Rb binding and phosphorylation of E7 by CKII are independent activities which are required for efficient transformation by E7 and that these activities correlate directly with the relative oncogenic potential of these viruses.

Previous experiments have demonstrated that transcription of the human c-fos oncogene is activated through the action of the 289-amino acid adenovirus E1A gene product. In this study we have utilized a series of c-fos promoter deletion and substitution mutants to define regulatory sequences that allow the induction by E1A. Although the deletion of upstream promoter sequences has varying degrees of effect on overall promoter activity, these deletions retain inducibility by E1A. This includes the deletion of the serum response element and two elements that bind the ATF transcription factor. In fact, a c-fos promoter deleted to position -53, which leaves the TATA element but no other known functional element, retains inducibility, indicating a role for the TATA element in E1A control. Indeed, substitution of the c-fos TATA element (TATAA) with a TATA sequence from the simian virus 40 early promoter (TATTTAT) abolishes E1A inducibility; this promoter does retain responsiveness to cAMP induction, however, demonstrating that this TATTTAT substitution is functional. We conclude that the E1A-dependent activation of c-fos transcription is mediated through an effect on a TATA-binding protein that has specificity for the TATAA sequence.

It has been shown previously that the adenovirus 2 (Ad2) E1A proteins repress activation of transcription by the SV40, polyomavirus and immunoglobulin gene enhancers. Here, we demonstrate that the repression of the SV40 enhancer is not specifically mediated by one of its constituent enhansons and/or proto-enhancers, but that each is subject to repression individually. This inhibitory effect of the E1A proteins is also observed with the AP-1 factor-binding enhansons from the polyomavirus and human metallothionein enhancers, and the MHC class I gene H-2Kb enhanson, which binds the KBF1/H2TF1/TC-IIB protein. Repression by the E1A gene products may, in fact, extend to all enhancer trans-activators, because the transcriptional activities of nuclear receptors (e.g., the estrogen and glucocorticoid receptors), of the yeast enhancer factor GAL4 expressed in HeLa cells, and of chimeric trans-activators (such as GAL-VP16) are all similarly inhibited. The E1A protein domains 2 and 3, including the acidic amino acid stretch that has been shown previously to be necessary for E1A-mediated trans-activation, are not required for repression. These results indicate that the amino-terminal region of the protein, which contains domain 1, plays a crucial role in repression, possibly by interfering in the transcriptional activation process at a step common to all trans-acting enhancer factors.

The hybrid plasmid pK4 containing the early genes of the simian virus SV-40, under the control of the adenovirus type 5 E1a promoter, was introduced into the multipotent embryonal carcinoma (EC) 1003. Expression of the SV-40 oncogenes was observed at the EC cell stage, and this allowed the derivation of immortalized cells corresponding to early stages of differentiation. Among the immortalized mesodermal derivatives obtained, one clone, C1, is committed to the osteogenic pathway. C1 cells have a stable phenotype, synthesize type I collagen, and express alkaline phosphatase activity. Although immortalized and expressing the SV-40 T antigen, the cells continue to be able to differentiate in vivo and in vitro. In vivo, after injection into syngeneic mice, they produce osteosarcomas. In vitro, the cells form nodules and deposit a collagenous matrix that mineralizes, going to hydroxyapatite crystal formation, in the presence of beta-glycerophosphate. This clonal cell line, which originates from an embryonal carcinoma, therefore differentiates into osteogenic cells in vivo and in vitro. This immortalized cell line will be useful in identifying specific molecular markers of the osteogenic pathway, to investigate gene regulation during osteogenesis and to study the ontogeny of osteoblasts.

This chapter describes a novel strategy that simplifies the generation and production of adenovirus type 5 (Ad5) mutants carrying defined mutations in early transcription units 1 (E1) and 4 (E4). The strategy involves three recombinant plasmids containing E1 (pE1-1235), E4 (pE4-1155), or the wild-type genome that lacks a portion of E3 (pH5pg4100). To generate recombinant viruses, mutations are first introduced into pE1- and/or pE4-transfer plasmids by site-directed mutagenesis. The mutagenized constructs are then ligated into plasmid pH5pg4100 containing the Ad backbone by direct cloning. Infectious viral DNAs are released from the recombinant plasmids by PacI-digestion and transfected into the complementing cell lines 293 or W162, and viral progeny are isolated and amplified. The advantages of this strategy are multiple: all cloning steps are carried out in Escherichia coli, and any genetic region of the viral E1 and/or E4 transcription units can be specifically modified or deleted. Moreover, foreign genes can be introduced into the E1 and/or E4 regions, and expression of viral or therapeutic genes can be controlled by cell-type specific and/or inducible promoters.

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

Invasiveness and metastatic potential are the two most important properties defining malignancy. The adeno-virus E1A (Ad-E1A) gene has a dual effect as a proliferative gene and as a tumor-suppressor gene, decreasing tumor growth and the metastatic potential of malignant cells. In order to study genes related with the antimetastatic effect of Ad-E1A in human cells, we performed a microarray analysis using OncoChiptrade mark. In three independent experiments, NIH3T3, IMR90 and MDA MB 435 cells were infected with pLPC retroviruses carrying the adenovirus 12S E1A gene or the GFP gene. We analyzed cDNA expression by using the CNIO OncoChipTM, a cDNA microarray containing a total of 6386 genes represented by 7237 clones. uPA, uPAr, tPA, PAI-1 and PAI-2 were also studied at RNA and protein levels. Microarrays of cDNA expression, RT-PCR and Western blot performed in IMR90 E1A-expressing cells showed downregulation of uPA, uPAr, tPA, PAI-1 and upregulation of PAI-2. These results were confirmed in NIH3T3 and MDA MB 435 breast carcinoma cells, with PAI-2 upregulation by RT-PCR and Western blot. In addition, zymographic analysis demonstrated that E1A expression greatly reduced the gelatinase activity of the pro-MMP2 and -MMP9 proteins. We propose that adenovirus E1A may orchestrate the expression of most members of the urokinase-plasminogen activation system, downregulating potentially invasive genes and upregulating PAI-2, which is associated with a better prognosis in human tumors.

Adenovirus (Ad) vectors are of utility for many therapeutic applications. Strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity employing fiber modifications allowing genetic incorporation of targeting motifs. In this regard, single chain antibodies (scFv) represent potentially useful agents to achieve targeted gene transfer. However, the distinct biosynthetic pathways that scFv and Ad capsid proteins are normally routed through have thus far been problematic with respect to scFv incorporation into the Ad capsid. Utilization of stable scFv, which also maintain correct folding and thus functionality under intracellular reducing conditions, could overcome this restriction. We genetically incorporated a stable scFv into a de-knobbed, fibritin-foldon trimerized Ad fiber and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of cells. We have shown that the scFv employed in this study retains functionality and that stabilizing the targeting molecule, per se, is critical to allow retention of antigen recognition in the adenovirus capsid-incorporated context.

In p53-dependent apoptosis in response to genotoxic and hypoxic stress, a fraction of induced wild-type p53 rapidly translocates to mitochondria, triggering a rapid first wave of mitochondrial membrane permeabilization and apoptosis that is later fortified by the transcriptional program of p53. However, whether this direct mitochondrial program also occurs upon oncogenic stress is unknown. In normal cells, oncogenic signals can induce a p53-dependent fail-safe mechanism to counter uncontrolled proliferation by engaging p53-dependent apoptosis. To address whether mitochondrial p53 contributes to oncogene-induced fail-safe apoptosis, p53 translocation was determined in primary human epithelial and endothelial cells overexpressing c-Myc, E1A or E2F1. Serum starvation of these cells, but not of control cells, triggered rapid p53 accumulation at mitochondria, accompanied by cytochrome c and SMAC release and followed by apoptosis. Our data establishes the contribution of the transcription-independent mitochondrial p53 pathway to apoptosis of primary cells in response to deregulated oncogenes.

First-generation adenovirus (Ad) gene therapy vectors deleted for the E1A, E1B, and E3 regions and carrying foreign genes under the control of strong foreign promoters induce high-level innate inflammatory responses within the first 24 hrs after transduction. Both uptake of the capsid and expression of gene products encoded by the vector contribute to the innate inflammatory response. Natural infections by Ad are frequently asymptomatic, suggesting that Ad has potent methods of inhibiting inflammation. The inability of Ad vectors to counter inflammatory responses suggests that the products of the Ad genes deleted in vector construction play critical roles in inhibiting these responses. Genetic analysis of the roles of Ad early region gene functions in vivo demonstrated that a virus made replication-incompetent by deletion of the preterminal protein gene and deleted for the transcriptional activation function of E1A effectively inhibits the innate inflammatory processes induced by Ad vectors. The mechanism(s) by which the Ad early region proteins inhibit inflammation is complex, as certain early region proteins can promote as well as inhibit inflammation, depending on the genetic context of the virus. Understanding of the roles of the Ad gene products in the induction and inhibition of innate inflammatory functions offers potential for the development of non-inflammatory vectors as well as for understanding of the mechanisms by which inflammation is regulated.

We identified a bifunctional regulatory element located between nt 374 and 431 upstream of TATA box of porcine adenovirus (PAV) 3 E1A promoter. Deletion of the element dramatically reduced the steady-state level of E1A mRNA, but increased that of E1B, which lies immediately downstream of E1A. The mutant virus displayed defective replication at early times of infection, but replicated nearly as efficiently as wild-type PAV-3 at late times of infection. This defect was complemented with coinfecting wild-type virus in a mixed infection. The results indicated that the upstream activation sequences (UAS) of E1A overlap the upstream repression sequences (URS) of E1B, although both transcription units are transcribed from different promoters.

We examined the transcriptional organization of fowl adenovirus 9 (FAdV-9) and analyzed temporal transcription profiles of its early and late mRNAs. At least six early and six late transcriptional regions were identified for FAdV-9. Extensive splicing was observed in all FAdV-9 early transcripts examined. Sequence analysis of the cDNAs representing the early proteins identified untranslated leader sequences, precise locations of splice donor and acceptor sites, as well as polyadenylation signals and polyadenylation sites. A unique characteristic, compared to other adenoviruses, was the detection by RT-PCR of multiple transcripts specific for each of five late genes (protein III, pVII, pX, 100K, and fiber), suggesting that FAdV-9 late transcripts undergo more extensive splicing than reported for other adenoviruses.

Adenoviruses (Ads) are endemic in the human population and the well-studied group C Ads typically cause an acute infection in the respiratory epithelium. A growing body of evidence suggests that these viruses also establish a persistent infection. The Ad genome encodes several proteins that counteract the host anti-viral mechanisms, which function to limit viral infections. This review describes the adenovirus immuno-regulatory proteins and how they function to block apoptosis of infected cells. In addition to facilitating the successful completion of the viral replication cycle and spread of progeny virus, these functions may help maintain the virus in a persistent state.

Adenoviral vectors are promising tools for pulmonary vascular gene transfer. In first generation vectors, the viral E4 region is preserved (E4+ Ad), but E4 is deleted in second generation vectors (E4- Ad). These vectors were compared for their toxicity in human endothelial cells in terms of apoptosis and necrosis. Infection with E4+ Ad vectors reduced whereas E4- Ad vectors enhanced apoptosis under normal culture conditions. Furthermore, E4+ Ad protected against apoptosis induced by growth factor deprivation, while E4- Ad enhanced apoptosis triggered by ceramide. Ad vectors containing different E4 open reading frames, alone or in different combinations, showed similar effects to E4- Ad, leaving the viral genes that might be responsible for reducing apoptosis unidentified at the present time. As previously observed with E4+ Ad devoid of transgene, E4+ Ad carrying beta-galactosidase or green fluorescent protein under the control of either the RSV or CMV promoter also reduced apoptosis triggered by growth factor deprivation. In contrast, E4+ Ad containing a CFTR expression cassette did not reduce apoptosis, and E4- Ad with CFFR showed increased toxicity. We conclude that Ad vectors may have important effects on the control of apoptosis in transfected cells, depending on the residual expression of viral genes. This effect can be complicated by the action of transgene expression on cell survival.

Adenoviruses have proved to be excellent tools for gaining insight into the regulation, and deregulation, of the mammalian cell cycle. With the widespread clinical use of gene therapy fast approaching, there comes a need for a better understanding of how the cell death process is regulated. A greater understanding will allow the development of therapeutic approaches that both maximise transgene expression while minimising cytotoxicity to the target cell. Consequently, much adenovirus research has centered on understanding the mechanisms governing adenovirus induced cell death or apoptosis. This review discusses recent advances in the field of adenovirus cell death regulation and evaluates the roles of implicated gene products and their respective data. The data suggest the existence of multiple virus gene products involved in cell death regulation and point towards several distinct, yet related, cell death pathways. A discussion of the shortcomings of current adenoviral research, along with a proposed model based upon the data is also given.

MHC class I molecules assemble with peptides in the endoplasmic reticulum (ER). To ensure that only peptide-loaded MHC molecules leave the ER, empty molecules are retained by ER-resident chaperones, most notably the MHC-specific tapasin. ER exit of class I MHC is also controlled by viruses, but for the opposite purpose of preventing peptide presentation to T cells. Interestingly, some viral proteins are able to retain MHC class I molecules in the ER despite being transported. By contrast, other viral proteins exit the ER only upon binding to class I MHC, thereby rerouting newly synthesized class I molecules to intracellular sites of proteolysis. Thus, immune escape can be achieved by reversing, inhibiting or redirecting the chaperone-assisted MHC class I folding, assembly and intracellular transport.

The p107 protein and the retinoblastoma protein (RB) both bind specifically to two viral oncoproteins, the SV40 T antigen (T) and adenoviral protein E1A (E1A). Like RB, p107 contains a segment (the pocket) that, alone, can bind specifically to T, E1A, and multiple cellular proteins. Cyclin A bound to the p107 pocket, but not the RB pocket. Although both pockets contain two, related collinear subsegments (A and B), the unique sequence in the p107 pocket that occupies the space between A and B is required for the interaction with cyclin A.

THE myc oncogene is functionally similar to adenovirus E1a in its ability to collaborate with activated ras oncogenes to transform primary fibroblasts1,2. The transforming functions of E1a and myc have been mapped to two distinct regions in each protein3,4. I investigated the functional similarities between Ela and myc by constructing E1a/myc chimaeras to discover whether the individual transforming domains of E1a could complement individual myc-transforming domains. Transformation assays in rat embryo fibroblasts demonstrated that the N-terminal transforming domain of E1a (CR1; ref. 5) could complement the C-terminal transforming domain of myc in cis, and that the reciprocal chimaera (N-terminal myc/C-terminal Ela) was also active. Chimaeras constructed using domains from transformation-defective mutants of either E1a or myc were inactive, indicating that both E1a and myc domains contribute to function. These experiments suggest that transformation by myc and E1a may involve interactions with common substrates.

PURPOSE: Adenovirus type 34 belongs to adenovirus subgenus B. The prototype virus of adenovirus 34 was isolated from a renal transplant recipient. However, no case of acute conjunctivitis caused by adenovirus 34 has been reported. Recently, we encountered two cases of acute follicular conjunctivitis in which adenovirus 34 was isolated. METHODS: The clinical isolates were identified by the standard neutralization test. The sequences of seven hypervariable regions in the hexon protein of these cases were compared with those of several prototype strains of adenovirus subgenus B. RESULTS: The cases were middle-aged, 34 and 41 years old, and male, and they exhibited moderate conjunctivitis with upper respiratory tract symptoms. Isolates from cell culture were identified as adenovirus 34 by NT. The mean homology rate (percentage of total number of coincident amino acids in the total length of amino acids in seven hypervariable regions) between clinical isolates and the adenovirus 34 prototype was 96.5%; in contrast, those between clinical isolates and the prototypes of adenovirus 11, adenovirus 14, and adenovirus 35 were 55.6%, 66.7%, and 57.9%, respectively. The results of conventional serotyping by neutralization test were confirmed by these values. CONCLUSIONS: These results indicate that adenovirus 34 may induce acute conjunctivitis in immunocompetent subjects and that special attention should be paid to adenovirus 34 as a causative agent for adenoviral conjunctivitis.

A recombinant adenovirus with deleted E1 and E3, and E4-inactivated by replacing the E4 promoter with a synthetic promoter composed of a minimal TATA box and five consensus yeast GAL4-binding site elements was developed and used to express the human tumor suppresser gene p53. The toxicity and immunogenicity of this vector and vector-mediated p53 gene expression in vivo were studied in immunocompetent C3H and C57BL/6 mice. Expression of the late viral gene product, hexon protein, was observed in C3H and C57BL/6 mice injected with E4 wild-type adenovirus constructs Adv-cmv-beta-Gal (BG), Adv-cmv-hp53 (WT), and empty E1- vector Adv-E4 (EW) 3 to 28 days after injection, but was undetectable in mice treated with E4 modified empty E1- vector Adv-GAL4 (EG) or Adv-cmv-hp53-GAL4 (G4). Expression of the p53 gene was observed in both WT- and G4-injected C3H and C57BL/6 mouse livers from days 3 to 28. Ten weeks after injection, p53 gene expression was still detected in G4-treated C57BL/6 mice at similar levels, but was not detectable in WT-treated mice. Vector-induced liver toxicity was evaluated by analyzing serum transaminases (SGOT and SGPT) activities. In all cases, SGOT and SGPT activities were markedly decreased in EG-treated C3H and C57BL/6 mice compared with those in EW-treated mice on days 3, 7 and 14 after injection. In C57BL/6 mice, the total anti-adenoviral CTL activities were two- to three-fold higher in animals treated with EW vector than in those treated with EG vector. These results suggest that inactivation of the E4 promoter efficiently diminished the viral replication and the late viral gene expression, reduced host immune response and consequently reduced toxicity and prolonged the duration of transgene expression in vivo.

To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C., Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R., and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex. In the latter compartments, CD8-E19 colocalizes with beta-coat protein (COP) (COPI component) and shows the higher density of labeling. Conversely, about 90% of the total CD8-KDEL protein is localized in clusters on the endoplasmic reticulum, where significant co-localization with Sec-23p (COPII component) is observed, and unglycosylated and initially O-glycosylated forms apparently constitute a single pool. Altogether, these results suggest that KKXX and KDEL retrieval motifs have different topological effects on theirs own at steady state: the first results in a specific enrichment in the intermediate compartment and cis-Golgi complex, and the latter dictates residency in the endoplasmic reticulum.

Proteins that activate genes are quite disparate in character; in particular, some work 'universally' and others do not. A simple model can accommodate most of the recently published results.

Adenoviruses often cause epidemic keratoconjunctivitis. The infection is highly contagious, often leads to corneal opacities, and is therefore to be timely diagnosed and properly treated. The authors propose a method for rapid diagnosis of adenovirus infection based on examination of a scraping off the conjunctiva by indirect immunofluorescence with polyvalent fluorescent rabbit serum and offer recommendations on the use of this method. Using this technique, adenovirus antigen was detected in 14 (61%) out of 23 patients with acute follicular conjunctivitis and in 19 (95%) out of 20 patients with keratoconjunctivitis. Despite clinical cure after antiviral therapy, the antigen persisted for 1 month in 22-30% patients and in some patients for 3 months (as a rule, it was in patients treated too late or ineffectively or after corticosteroids). That is why maintenance antivirus therapy is recommended for 2-3 months after clinical cure.

We have recently shown that the adenovirus type 5 E4orf6 protein interacts with the cellular tumor suppressor protein p53 and blocks p53 transcriptional functions. Here we report that the E4orf6 protein can promote focus formation of primary rodent epithelial cells in cooperation with adenovirus E1A and E1A plus E1B proteins. The E4orf6 protein can also inhibit p53-mediated suppression of E1A plus E1B-19kDa-induced focus formation. Mutant analysis of the E4orf6 protein demonstrates that these activities correlate with the ability of the adenovirus protein to relieve transcriptional repression mediated by the carboxyl-terminal region of p53 in transient transfection assays. We further demonstrate that expression of wild-type E4orf6 correlates with a dramatic reduction of p53 steady-state levels in transformed rat cells. Our data demonstrate that adenovirus type 5 encodes two different proteins, E1B-55kDa and E4orf6, that bind to p53 and contribute to transformation by modulating p53 transcriptional functions.

The origin of human adenovirus type 4 (Ad4), an important pathogen and candidate vaccine vector, has been the subject of speculation. Ad4 is unusual among adenoviruses, because it is the single known serotype of subgroup E. Some biological and biochemical properties of Ad4 resemble those of serotypes from subgroups B and C. The length of Ad4 fiber is intermediate between that of subgroup B and C fibers. We sequenced the Ad4 fiber gene, locus of the determinant(s) of adenovirus serotype. The number of repeating DNA sequence motifs in the shaft domain of the Ad4 fiber gene is consistent with its reported length. Regional phylogenetic analysis of Ad4 was undertaken, comparing DNA sequences of early genes and fiber genes from representative adenoviruses. The Ad4 fiber gene has close phylogenetic relationship to subgroup C fiber genes. This is in distinct contrast to the closer relationship of Ad4 to subgroup B adenoviruses in early gene sequences, distributed across the left 70% of the viral genome. We propose that Ad4 originated by recombination of genomes resembling contemporary subgroup B and subgroup C adenoviruses. This event may have occurred so recently that divergence of subgroup E serological determinants has yet to be observed.

The haemagglutinin—esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombination between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [3H]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypeptide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haemagglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recombinant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo.

The 12 S and 13 S E1 A cDNAs from both the Adenovinus (Ad) nononcogenic type 5 and the oncogenic type 12 were overexpressed in an insect cell/baculovirus system. Upon infection of Spodoptera frugiperda cells, the production of E1A proteins reached a level of about 15 μg/10 6 Cells. The E1A proteins are highly soluble and apparently are processed authentically. They are readily recognized by various antibodies and display phosphorylation patterns similar to those of E1 A proteins synthesized in mammalian cells. Single-step immunoaffinity chromatography was used to purify the Ad5 E1A proteins to near homogeneity under nondenaturing conditions. The Ad5 and Ad12 E1 A proteins are able to form complexes with the retinoblastoma susceptibility gene product (Rb) and other cellular proteins. Interestingly, the presence of a cellular extract seems to be a prerequisite for association between highly purified E1A and Rb polypeptides.

Members of the helix-loop-helix (HLH) family of proteins bind DNA and activate transcription as homo- and heterodimers. Myogenin is a muscle-specific HLH protein that binds DNA in vitro as a heterodimer with several widely expressed HLH proteins, such as the E2A gene products E12 and E47. We describe a method for detection of protein-protein interactions among HLH proteins in vivo in which dimerization through the HLH motif reconstructs a hybrid transcription factor containing the DNA-binding domain of yeast GAL4 linked to one HLH motif and the activation domain of VP-16 linked to another. We have used this assay to investiagate whether myogenin forms homomeric or heteromeric complexes in vivo and to determine whether growth factors and oncogenes that inhibit myogenesis influence myogenin's ability to dimerize. The results show that myogenin heterodimerizes with E12 and E47 in vivo, but it does not homodimerize to a measurable extent. Peptide growth factors, as well as the immediate early gene products c-Jun, v-Fos, and c-Myc, inhibit the activity of myogenin through a mechanism independent of its association with E2A products.

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat β-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.

Early region 4 (E4) of mouse adenovirus type 1 was analyzed by Northern blotting, cDNA sequencing, and S1 nuclease protection and primer extension assays. The transcription map of this region was dissimilar to the consensus human adenovirus E4 transcription map in which all transcripts have identical 5' and 3'-terminal sequences. Seven classes of mouse adenovirus type 1 mRNAs were identified; all shared the same 3' end. Three classes of unspliced mRNAs differed at their 5' start sites, two classes of spliced transcripts differed in the locations of their splice acceptors, and two classes of spliced messages differed in their splice donors and acceptors. From the structure of the various transcripts, translational products were predicted. In addition to a predicted polypeptide with similarity to the human adenovirus 2 E4 34K protein previously identified (A. O. Ball, C. W. Beard, P. Villegas, and K. R. Spindler, 1991, Virology 180, 257-265), two open reading frames with similarity to human adenovirus 2 E4 open reading frames 2 and 3 were found.

Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis.

The protein product of the retinoblastoma susceptibility gene, p110RB1, is a nuclear phosphoprotein [W.H. Lee, J.Y. Shew, F.D. Hong, T.W. Sery, L.A. Donoso, L.J. Young, R. Bookstein, and E.Y. Lee, Nature (London) 329:642-645, 1987] with properties of a cell cycle regulator (K. Buchkovich, L.A. Duffy, and E. Harlow, Cell 58:1097-1105, 1989; P.L. Chen, P. Scully, J.Y. Shew, J.Y. Wang, and W.H. Lee, Cell 58:1193-1198, 1989; J.A. DeCaprio, J.W. Ludlow, D. Lynch, Y. Furukawa, J. Griffin, H. Piwnica-Worms, C.M. Huang, and D.M. Livingston, Cell 58:1085-1095, 1989; and K. Mihara, X.R. Cao, A. Yen, S. Chandler, B. Driscoll, A.L. Murphree, A. TAng, and Y.K. Fung, Science 246:1300-1303, 1989). Although the mechanism of action of p110RB1 remains unknown, several lines of evidence suggest that it plays a role in the regulation of transcription. We now show that overexpression of p110RB1 causes repression of the adenovirus early promoter EIIaE and the promoters of two cellular genes, c-myc and RB1, both of which contain E2F-binding motifs. Mutation of the E2 element in the c-myc promoter abolishes p110RB1 repression. We also demonstrate that a p110RB1 mutant, which is refractory to cell cycle phosphorylation but intact in E1a/large T antigen-binding properties, represses EIIaE with 50- to 80-fold greater efficiency than wild-type p110RB1. These data provide evidence that hypophosphorylated p110RB1 actively represses expression of genes with promoters containing the E2F-binding motif (E2 element).

Functional analysis of human papillomavirus type 16 E7 protein by complementation with adenovirus E1A mutants in baby rat kidney cells has shown that the retinoblastoma gene product (RB)-binding region of E7 can substitute in trans for that of E1A. An N-terminal E7 mutant was unable to complement an E1A mutant unable to bind p300, indicating that the two mutants were defective for functionally equivalent activities. E7 proteins with mutations within the RB-binding region were also unable to complement either the non-p300-binding E1A mutant or the N-terminal E7 mutant, suggesting that these mutations affect more than just RB binding.

A t(17;19) chromosomal translocation in early B-lineage acute leukemia was shown to result in chimeric transcripts that contain sequences from the E2A basic helix-loop-helix transcription factor gene on chromosome 19, fused to sequences from a previously unidentified gene (HLF) on chromosome 17 that encodes a hepatic leukemia factor. The chimeric protein consisted of the amino-terminal transactivation domain of E2A linked to the carboxyl-terminal basic region-leucine zipper domain of HLF. HLF was normally expressed in liver and kidney, but not in lymphoid cells, and was found to be closely related to the leucine zipper-containing transcription factors DBP (albumin D-box binding protein) and TEF (thyrotroph embryonic factor), which regulate developmental stage-specific gene expression.

ORIGINALLY E2F sites were identified as elements in the promoters of adenovirus early genes that are necessary for activation of these genes by the early protein E1a (ref. 1). E2F promoter elements have been shown to be important for transcriptional activation of several genes critical for progression through the cell cycle2–4. During the G1 phase of the cell cycle, the E2F protein forms a complex with the cell-cycle protein Rb (ref. 5) and it has been suggested that this binding of Rb to E2F inactivates E2F (ref. 5). Here we show that Rb-E2F is an active complex that, when bound to the E2F site, inhibits the activity of other promoter elements and thus silences transcription. We propose that the ability of this complex to inhibit transcription is integral to the function of Rb and provide evidence that E2F is a positive element in the absence of an active form of Rb. It has been shown that binding of Rb to E2F depends on the phosphorylation state of Rb (only the underphosphorylated form binds)5 and that the phosphorylation state of Rb changes during progression through the cell cycle6,7. We therefore suggest that the E2F site alternates between a positive and negative element with the phosphorylation/dephosphorylation cycle of Rb. This cyclic activity may be responsible for activating and then inhibiting genes during the cell cycle.

Four murine cellular tumor models expressing various combinations of oncogenes (SV40 large T and v-Ha-ras, SV40 large T and v-src, SV40 large T and neu, adenovirus E1A and v-Ha-ras) induce sarcoma when they are inoculated s.c. into the DBA/2 syngenic mice. The metastatic patterns, distribution and fate of these tumor cells transplanted by two different routes into syngenic DBA/2 mice have been studied. All the tumor cell lines except E1A-ras, induce massive overt artificial metastases principally in the lung after i.v. injection. In s.c. tumor-bearing mice, a few resting cells colonize the lung as micrometastases. When removed from this tissue context and injected s.c. these cells regain their proliferative potential and grow as local tumors which again give rise to occult pulmonary micrometastases. © 1992 Wifey-Liss, Inc.

We have characterized the biosynthesis and processing of a 91 amino acid hydrophobic integral membrane protein encoded by human group C adenoviruses which down-regulates the EGF receptor (Carlin, C. R., Tollefson, A. E., Brady, H. A., Hoffman, B. L., and Wold, W. S. M. (1989) Cell 57, 135-144). Previous studies have shown that two immunologically related proteins are produced in vivo, a 13.7-kDa protein encoded by E3 message f and a 11.3-kDa protein derived from 13.7 kDa by proteolysis (Hoffman, B. L., Ullrich, A., Wold, W. S. M., and Carlin, C. R. (1990) Mol. Cell. Biol. 10, 5521-5524; Tollefson, A. E., Krajcsi, P., Yei, S., Carlin, C. R., and Wold, W. S. M. (1990) J. Virol. 64, 794-801). We report here that the 13.7- and 11.3-kDa proteins form intermolecular disulfide bonds cotranslationally at Cys-31 and tend to migrate as high molecular weight aggregates under nonreducing conditions. Both proteins are also present at the cell surface, as evidenced by specific immunoprecipitation from intact monolayers enzymatically labeled with 125I. Moreover, an antiserum specific for a putative extracellular epitope recognizes the same viral proteins as antibodies directed against a C-terminal synthetic 15-mer. The 13.7- and 11.3-kDa proteins are detected at early time points during pulse-chase radiolabeling of infected cells, do not undergo any further changes in molecular weight, and focus at their predicted isoelectric points (7.4 and 7.2, respectively). Identical results are obtained in stable transfectants constitutively expressing only 13.7 and 11.3 kDa, suggesting that biosynthesis and processing is not dependent on other viral proteins. These results have been incorporated into a computer-based model to predict the orientation of 13.7 and 11.3 kDa in the lipid bilayer. This model provides a basis for testing predictions regarding the topology of the viral proteins, as well as putative interactions with heterologous proteins in the microenvironment of the plasma membrane that cause down-regulation of the epidermal growth factor receptor.

Replication of human adenovirus type 12 DNA is blocked in abortively infected baby hamster kidney cells. The activity and accumulation of adenovirus 12 DNA polymerase is equivalent in infected hamster and human cell extracts. However, the accumulation of adenovirus type 12 DNA-binding protein is approximately 120-fold lower in extracts from infected hamster cells when compared to infected permissive human cells. This difference in accumulation is not due to replication of viral DNA during productive infection, since this difference is observed in the presence of hydroxyurea. The DNA-binding protein from infected hamster cells retains the ability to bind denatured DNA-cellulose. An adenovirus 5 early region 1 transformed hamster cell line competent to complement the adenovirus 12 DNA replication defect also stimulates accumulation of the DNA-binding protein even when the cells are treated with hydroxyurea. Thus, the reduced expression of the viral DNA-binding protein may play a role in the mechanism of abortive infection of hamster cells by adenovirus 12.

The E1a gene of group C adenoviruses is one of the most studied transforming genes of DNA tumor viruses. These transforming genes have been conventionally considered as dominant oncogenes since they transform cells in vitro and many of the resulting transformed cell lines induce tumors in experimental animals. It now appears that, in addition to its well-known transforming activities, E1a possesses activities which suppress transformation, tumorigenesis and malignant progression (metastasis) of tumor cells. Thus, E1a appears to meet the definition of both a dominant oncogene and a tumor-suppressor gene.

Hybrid adenovirus type 12 (Ad12)/Ad5 E1A genes were constructed by homologous recombination in Escherichia coli, a technique which offers several advantages over conventional mutagenesis for genetic analysis of proteins. In particular, functional differences between the proteins can be mapped by correlating the replacement of specific sequences with the acquisition of new properties, and there is no requirement for common unique restriction sites or polymerase chain reaction strategies to construct the hybrids. Recombinant adenoviruses expressing these hybrid E1A proteins were capable of replicating efficiently in HeLa cells, with the exception of one construct which contained a hybrid transactivation domain. The transforming activity of the hybrid E1A constructs was assayed by DNA transfection of primary baby rat kidney cells. Plasmids containing Ad12 E1 were approximately 20-fold less efficient at transformation than those with E1 of Ad5, and it was found that two regions in exon 1 of E1A mediate this difference. No differences were found in the abilities of any hybrid E1A proteins to bind to cellular proteins previously determined to be important for transformation by E1A.

Two regions near the amino terminus of the adenovirus E1A protein, which were first identified by sequence conservation among various adenovirus serotypes, have been shown by genetic studies to be essential for E1A-mediated transformation. These same regions are also required for interaction with a number of cellular proteins, including the retinoblastoma protein (pRB). Using synthetic peptides corresponding to portions of these conserved regions, we show that each region can bind independently to pRB. These interactions were observed in both competition and binding assays. In both types of assay, region 2 peptides (E1A amino acids 115 to 132) bound pRB with higher affinity than did region 1 peptides (E1A amino acids 37 to 54), while a peptide combining region 1 and 2 sequences consistently provided the highest-affinity interaction. Cross-blocking experiments using region 1 peptides and region 2 peptides suggested that these two regions of E1A make distinct contacts with pRB. These data support the notion that the pRB-binding domain of E1A contains at least two functional elements.

Wilms tumor is a pediatric kidney cancer that has been linked to the inactivation of a tumor-suppressor gene at chromosome locus 11p13. The WT1 gene, mapping to this locus, is developmentally regulated in the kidney and encodes a putative transcription factor that has been shown to be mutated in Wilms tumor specimens. We have suggested that one such altered product of the WT1 gene may be capable of trans-dominant suppression, since the mutated allele was found to be coexpressed with the wild-type allele in a sporadic Wilms tumor. We therefore tested the ability of this mutant WT1 allele, containing an in-frame deletion within the DNA-binding domain, to transform primary baby rat kidney cells. The mutant WT1 gene was found to cooperate with the adenoviral E1A gene in transforming baby rat kidney cells, as demonstrated by growth in soft agar and tumorigenicity in nude mice. The wild-type WT1 gene in all of its alternatively spliced forms neither suppressed E1A-induced focus formation nor cooperated with E1A. Our results indicate that impairment of DNA binding of the WT1 tumor-suppressor gene product can result in a dominant negative mutation.

The adenovirus E3-14.5K protein is a cytoplasmic integral membrane protein that functions in concert with the E3-10.4K protein to down-regulate the epidermal growth factor receptor and to prevent tumor necrosis factor cytolysis in adenovirus-infected cells. The 14.5K protein migrates as multiple bands in SDS-PAGE, indicating that it undergoes post-translational modification. The 14.5K protein is known to be phosphorylated on serine. We show here that 14.5K can be metabolically labeled with [3H]glucosamine, that the label is labile to alkali, and that the SDS-PAGE band pattern is simplified in a cell line that is defective in O-glycosylation. Thus, 14.5K is O-glycosylated, probably at a single site in the NH2-terminal lumenal domain. The protein was not metabolically labeled with [3H]mannose, and its SDS-PAGE band pattern was not affected by tunicamycin treatment in vivo or endo F treatment in vitro; thus, 14.5K is not N-glycosylated. There was no evidence that the 10.4K protein is glycosylated, and the 10.4K protein was not required for glycosylation of 14.5K. Virtually all 14.5K molecules appear to contain the core disaccharide Gal beta 1-3GalNAc alpha 1-Ser/Thr which is commonly found on mucin-type O-glycoproteins, and neuraminidase digestion experiments indicated that this disaccharide contains terminal sialic acid.

The retinoblastoma tumor-suppressor gene encodes a 105-kDa nuclear phosphoprotein (RB) that can associate with DRTF-1 and E2F. These two transcription factors can recognize the same DNA motif in the adenovirus E2A promoter and can bind to it by themselves or in association with RB. In the present report, we describe the use of CASTing (cyclic amplification and selection of targets) to determine the consensus binding site of RB-containing complexes. An anti-human RB antibody was used to isolate RB-containing complexes formed after mixing nuclear extracts obtained from human diploid fibroblasts with a pool of random oligonucleotides flanked with polymerase chain reaction (PCR) primers. After the immunoselection, the DNA was isolated, amplified, mixed with fresh nuclear extract and reselected. After six CASTing cycles, the DNA was cloned and sequenced. We found that the highest affinity motifs recognized by RB-containing complexes are related to the E2F/DRTF-1 binding site and fall into three classes: TTTTCCCGCCAAAA, TTTTCCCGCCTTTTTT or TTTTCCCGCGCTTTTTT. Competition experiments revealed that these three classes are functionally equivalent to each other and to the E2F/DRTF-1 binding site in the adenovirus E2A promoter. Screening these sequences against a DNA database identified their presence in non-coding regions of many oncogenes, growth factor genes and in the RB gene itself.

One of the major E1A-associated cellular proteins is a 300-kDa product (p300) that binds to the N-terminal region of the E1A products. The p300 binding site is distinct from sequences involved in binding the retinoblastoma product and other E1A-associated cellular products such as p60-cyclin A and p107. p300 binding to E1A is linked genetically to the enhancer repression function of E1A and the other E1A-mediated gene-regulating functions as well as to the transforming functions of E1A. However, the biochemical properties of p300 have not yet been characterized. We report here that p300 has an intrinsic DNA-binding activity and shows a preferential affinity for specific DNA sequences. The sequences selectively bound by p300 are related to those of a series of enhancer elements that are recognized by NF-kappa B. The direct physical interaction of p300 with enhancer elements provides a biochemical basis for the genetic evidence linking the E1A-mediated enhancer repression function with the p300-binding activity of E1A.

A plasmid carrying the 5′-flanking region (– 1584 to + 47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5- to 9-fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of El A resides in a common region(s) of 13S and 12S El A products. The major target region of El A was mapped within the 68 base-pair region (-21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2P, although the upstream region (–83 to – 21) including ATF(CREB)-binding consensus had an additional effect in the transactivation.

The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of E1A expression. To study the mechanism of this E1A-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with E1A expression plasmids. The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary E1A-responsive elements for repression of the FN gene. Two GC boxes also contain a G10 stretch that is interrupted by the presence of an internal C residue. These sequences overlap with the Sp1 motif GGGCGG. Substitution of the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, completely abolished the E1A sensitivity of the promoter. Analysis of the E1A domains by using various E1A deletion mutants indicated that the domain for binding to the retinoblastoma susceptibility gene product (RB) is essential for efficient repression. These results suggest that the gene encoding a negative factor(s) binding to the three G-rich sequences in the FN promoter is repressed by RB in resting 3Y1 cells and derepressed by expression of E1A.

A plasmid carrying the 5'-flanking region (-1852 to +33 with respect to the transcription initiation site) of the mouse DNA polymerase beta gene fused with the chloramphenicol acetyltransferase (CAT) gene was cotransfected into mouse N18TG2 cells with adenovirus type 12 E1 genes-expressing plasmids. Expression of E1A gene products resulted in the elevation of the CAT expression by 3 to 7 folds, but that of E1B gene product was much less effective. RNase protection analysis revealed that the activation by E1A was at the transcription process. Both the 13S E1A and the 12S E1A activated the DNA polymerase beta gene promoter, indicating that the activation domain of E1A is in a common region(s) of 13S and 12S E1A products. The major target sequence of E1A was mapped within the 10 base pair-region (-30 to -20) of the DNA polymerase beta gene promoter, which overlapped with the palindromic sequence known as the ATF(CREB)/E4F-binding consensus. The results suggest that the palindromic sequence is essential for E1A-induced transcriptional activation of the mouse DNA polymerase beta gene.

THE cellular phosphoprotein p53 inhibits progression through the mammalian cell cycle1,2. Both p53 alleles are frequently mutated in human tumours3,4, indicating that p53 is a tumour suppressor. Recent studies have suggested that p53 functions as a transcrip-tional activator5–8, but the significance of this activity in cell-cycle control has not been established. The adenovirus 2 (Ad2) early IB (E1B) 55K protein binds to p53 in transformed cells9 and contributes to oncogenic transformation by Ad2 (refs 10-12). Here we report that mutants of E1B 55K and wild-type Adl2 E1B 54K proteins show a strong correlation between their ability to inhibit p53-mediated transcriptional activation and their ability to cooperate with adenovirus El A protein in the transformation of primary cells. These results indicate that p53 probably inhibits cell cycling by functioning as a transcription factor.