Search

A broad spectrum of genetic and molecular investigations carried out with group C, Ad2 and Ad5, and with group A, Ad12, have shown that early region1 (E1) gene products are sufficient for complete transformation of rodent cells in vitro by these viruses. During the past quarter century, the processes by which E1A proteins, in cooperation with E1B proteins, perturb the cell cycle and induce the transformed phenotype, have become well defined. Somewhat less understood is the basis for the differential oncogenicity of these two groups of viruses, and the processes by which the E1A proteins of Ad12 induce a tumorigenic phenotype in transformants resulting from infection of cells in vivo and in vitro. In this chapter we review previous findings and present new evidence which demonstrates that Ad12 E1A possesses two or more independent functions enabling it to induce tumors. One of these functions lies in its capacity to repress transcription of MHC class I genes, allowing the tumor cells to avoid lysis by cytotoxic T lymphocytes. We have shown that class I repression is mediated through increased binding of repressor COUP-TF and decreased binding of NF-kB to the class I enhancer. In addition to mediating immune escape, E1A also determines the susceptibility of transformants to Natural Killer (NK) cell lysis, and in this case, also, Ad12 transformants are not susceptible. By using Ad12 mutants containing chimeric E1A Ad12-Ad5 genes, point mutations, or a specific deletion, we have shown that the unique spacer region of Ad12 E1A is an oncogenic determinant, but is not required for transformation in vitro. Given that the E1A regions responsible for class I repression are first exon encoded, we have examined a set of cell lines transformed by these altered viruses, and have found that while they display greatly reduced tumorigenicity, they maintain a wildtype capacity to repress class I transcription. Whether the spacer contributes to NK evasion remains unresolved. Lastly, we discuss the properties of the Ad2/Ad5 E1A C-terminal negative modulator of tumorigenicity, and examine the effects on transformation, tumor induction and transformant tumorigenicity, when the Ad5 negative modulator is placed by chimeric construction in Ad12 E1A.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) is an orphan nuclear receptor that represses transcription of many genes. In adenovirus type 12 (Ad12) transformed cells, a high level of binding activity of COUP-TF to the major histocompatibility complex (MHC) class I enhancer correlates with the down-regulation of class I transcription, which, in turn, contributes to tumorigenesis. The mechanism by which COUP-TF represses transcription has yet to be elucidated. Here we show that COUP-TF represses transcription through its association with histone deacetylase. This was demonstrated using reciprocal binding assays that determined that the interaction between COUP-TF and histone deacetylase requires the COUP-TF C-terminal repression domain. Moreover, a histone deacetylase enzymatic activity was found to be associated with COUP-TF in Ad12-transformed cells. Transfection experiments further revealed that exogenous histone deacetylase facilitates transcriptional repression by COUP-TF. Also, supershift assays suggest that the transcriptional corepressor N-CoR, which is known to associate with histone deacetylases, is a part of the COUP-TF complex bound to the MHC class I enhancer R2 site. Finally, we provide evidence that inhibition of histone deacetylases relieves the repression of MHC class I expression in Ad12-transformed cells. Taken together these results support the notion that deacetylation of histones, mediated through COUP-TF, serves to down-regulate MHC class I transcription in Ad12-transformed cells., (Copyright 2000 Academic Press.)

Reduced cell surface levels of major histocompatibility complex class I antigens enable adenovirus type 12 (Ad12)-transformed cells to escape immunosurveillance by cytotoxic T lymphocytes (CTL), contributing to their tumorigenic potential. In contrast, nontumorigenic Ad5-transformed cells harbor significant cell surface levels of class I antigens and are susceptible to CTL lysis. Ad12 E1A mediates down-regulation of class I transcription by increasing COUP-TF repressor binding and decreasing NF-kappaB activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-kappaB in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-kappaB dimers reconstituted from denatured and renatured p50 and p65 subunits from Ad12- and Ad5-transformed cell nuclear extracts demonstrated that p50, and not p65, is responsible for the decreased ability of NF-kappaB to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-kappaB to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-kappaB binding was further demonstrated by showing that an NF-kappaB dimer composed of p65 and alkaline phosphatase-treated p50 from Ad5-transformed cell nuclear extracts could not bind to DNA. These results suggest that phosphorylation of p50 is a key step in the nuclear regulation of NF-kappaB in adenovirus-transformed cells.

The CR3 activation domain of the human adenovirus E1A protein stimulates transcription by forming protein-protein interactions with DNA sequence-specific binding factors and components of the TFIID complex. Here, we demonstrate that CR3 can complex with the extreme C-terminal 105 amino acids of the human TATA box binding-factor-associated protein, hTAF(II)135. Furthermore, the C-terminal region of hTAF(II)135 can block transcriptional stimulation from an E1A-inducible promoter in vivo. This ability of the C terminus of hTAF(II)135 to bind CR3 and to inhibit E1A-inducible activation is highly specific. These results demonstrate for the first time that a discrete fragment of a mammalian TBP-associated factor which targets a specific activator can impair the stimulation of transcription.

Diminished expression of major histocompatibility complex class I antigens on the surface of adenovirus type 12 (Ad12)-transformed cells contributes to their high tumorigenic potential by enabling them to escape immune recognition by cytotoxic T lymphocytes. This low class I antigen expression is due to a block in class I transcription, which is mediated by Ad12 E1A. Genetic analysis has shown that the class I enhancer is the target for transcriptional down-regulation. In this study, we show that the ability of the R1 element of the class I enhancer to stimulate transcription is greatly reduced in Ad12-transformed cells. The loss of functional activity by the R1 element was attributed to loss of binding by the NF-kappa B p50-p65 heterodimer. NF-kappa B binding appears to be blocked within the nucleus rather than at the level of nuclear translocation. Significantly, NF-kappa B binding activity could be recovered from the nuclear extracts of Ad12-transformed cells following detergent treatment, suggesting that the block is mediated through a nuclear inhibitor present in the Ad12-transformed cells. These results, taken together with the fact that the R2 element of the class I enhancer exhibits strong binding to the transcriptional repressor COUP-TF, suggest that the class I enhancer is globally down-regulated in Ad12-transformed cells.

The transcriptional down-regulation of the major histocompatibility complex (MHC) class I antigens in adenovirus type 12 (Ad12) transformed cells gives them the potential to escape immunosurveillance and to form tumors. The enhancer of the class I promoter is the target of transcriptional repression which is mediated by the E1A gene of Ad12. The R2 region within the class I enhancer acts as a negative element in Ad12-transformed cells and exhibits a stronger binding activity than is observed in nontumorigenic Ad5-transformed cells, which are not reduced in class I expression. The R2 element contains a nuclear hormone receptor half-site consensus sequence, AGGTCA, which is required for both the binding activity and the ability of R2 to act as a negative element in Ad12-transformed cells. In this study, we show that an orphan hormone receptor protein, COUP-TF, contributes to the differential R2 binding activity observed between Ad12- and Ad5-transformed cells. Additionally, COUP-TF was shown to bind as a dimer to the R2 element and to use the consensus AGGTCA as one half-site and its 3' flanking sequence as a probable second degenerate half-site. Since COUP-TF can act as a transcriptional repressor, we suggest that the higher COUP-TF binding activity to the R2 element in Ad12-transformed cells contributes to down-regulation of class I transcription and, consequently, tumorigenesis.

The cell growth-regulating properties of the adenovirus type 5 (Ad5) E1A oncogene correlate closely with the binding of the E1A products to specific cellular proteins. These proteins include the products of the retinoblastoma tumor susceptibility gene and a 300-kDa product, p300. pRB binds to E1A sequences that are highly conserved among the E1A products of various serotypes, while p300 binding requires sequences in the E1A amino terminus, a region that is not highly conserved. To help evaluate the roles of the E1A-associated proteins in cell growth control, we have compared the p300-binding abilities of the E1A products of Ad5 and of the more oncogenic Ad12 serotype. We show here that despite encoding a sequence that varies somewhat from the p300-binding sequences of Ad5 E1A, the Ad12 E1A products associate with p300 with an affinity similar to that of the Ad5 E1A products. Both the 12S and 13S splice products of Ad12 E1A, like those of Ad5 E1A, encode proteins able to associate with p300. Interestingly, though, both also give rise to prominent forms that are amino terminally modified and unable to associate with p300. This modification, at least in the 13S product, does not appear to diminish the affinity of this product for the retinoblastoma protein.

In transformed cells, the E1A gene of adenovirus type 12 (Ad12) represses transcription of class I genes of the major histocompatibility complex. The tumorigenic potential of Ad12-transformed cells correlates with this diminished class I expression. In contrast, the E1A gene of the nontumorigenic Ad5 does not affect class I expression. We show here that a transfected reporter chloramphenicol acetyltransferase plasmid driven by an H-2K promoter (-1049 bp) was expressed at much lower levels in Ad12- than in Ad5-transformed mouse cells. Analysis of mutant constructs revealed that only 83 bp of H-2 DNA, consisting of the enhancer juxtaposed to the basal promoter, was sufficient for this differential expression. Whereas the H-2 basal promoter alone was somewhat less active in Ad12-transformed cells, the H-2 TATA box itself did not appear to be important. The H-2 enhancer proved to be the principal element in Ad12 E1A-mediated repression, since (i) substitution of the H-2 enhancer by simian virus 40 enhancers overcame the repression, and (ii) when juxtaposed to either its native or heterologous basal promoters, the H-2 enhancer was functional in Ad5- but not Ad12-transformed cells. Mobility shift assays showed that there is a DNA-binding activity to the 5' site (R2 element) of the enhancer that is significantly higher in Ad12- than in Ad5-transformed cells. These results suggest that decreased class I enhancer activity in Ad12-transformed cells may, at least in part, be due to the higher levels of an enhancer-specific factor, possibly acting as a repressor.

In cells transformed by the highly oncogenic adenovirus type 12 (Ad12), the viral E1A proteins mediate transcriptional repression of the major histocompatibility class I genes. In contrast, class I transcription is not reduced in cells transformed by the nononcogenic Ad5. The decreased rate of class I transcription is, at least in part, the result of a reduced major histocompatibility complex class I enhancer activity in Ad12-transformed cells and correlates with an increase in the levels of a DNA-binding activity to the R2 element of the enhancer (R. Ge, A. Kralli, R. Weinmann, and R. P. Ricciardi, J. Virol. 66:6969-6978, 1992). Employing transient transfection assays, we now provide direct evidence that the R2 element can confer repression in Ad12- but not Ad5-transformed cells. Repression by R2 was observed only in the presence of the positive enhancer element R1 and was dependent on (i) the number of the R2 elements and (ii) the relative arrangement of R2 and R1 elements. The putative R2-binding repressor protein, R2BF, was similar in molecular weight and binding specificity to members of the thyroid hormone/retinoic acid (RA) receptor family. RA treatment abrogated the R2-mediated repression in Ad12-transformed cells and had no effect on the activity of R2/R1-containing promoters in Ad5-transformed cells. These results are consistent with the presence of an R2-binding repressor in Ad12-transformed cells. In the absence of RA, the repressor compromises enhancer activity by interfering with the activity of the positive cis element R1. RA treatment of Ad12-transformed cells may render the repressor inactive.

The gene of the human cytomegalovirus (HCMV) major envelope glycoprotein, gB, was cloned from the Towne strain and inserted into adenovirus type 5 downstream of the E3 promoter. The recombinant virus, Ad-gB, expressed antigenically related proteins of 58, 30, 25, and 23 kDa in A549 and MRC-5 cells; the 58-kDa protein had the same mobility as the native gB from HCMV-infected MRC-5 cells and virions. All four proteins were detected by a monospecific polyclonal antiserum and by a monoclonal antibody in immunoblot and immunofluorescence assays. Hamsters infected intranasally with live Ad-gB developed protein-specific and HCMV-neutralizing antibody. This study confirms the importance of gB in the generation of the neutralizing immune response to HCMV and demonstrates the potential of live adenoviruses as vaccine vectors.

Host-range mutants of adenovirus 5 that contain a defect in region E1A (0-4.5 units) fail to replicate in HeLa cells and to transform rodent cells. In HeLa cells, these mutants synthesize only the two RNAs from E1A that share the same 5' and 3' termini but differ in length by the amount of internal sequence removed by splicing. RNA from wild-type virus, selected by hybridization to DNA from region E1A, translates into polypeptides of Mr 51,000 and 48,000 that are highly acidic in isoelectric focusing gels. These acidic Mr 51,000 and Mr 48,000 polypeptides are encoded by the longer and shorter E1A RNAs, respectively. Two of the host-range mutants, H5hr1 and H5hr2, fail to synthesize the Mr 51,000 polypeptide but do produce the Mr 48,000 polypeptide and a novel polypeptide thought to be a truncated portion of the Mr 51,000 polypeptide. H5hr1 and H5hr2 are hypothesized to have termination codons in sequences found only in RNA encoding the Mr 51,000 polypeptide. This prediction is verified for H5hr1 by DNA sequence analysis. The other three host-range mutants (H5hr3-5) synthesize both acidic polypeptides and are predicted to be missense. These results strongly imply that the Mr 51,000 polypeptide, alone or in combination with the Mr 48,000 polypeptide, is needed to regulate expression of adjacent viral genes during the early phase of adenovirus infection.

The E3 regions of adenovirus types 2 and 5, respectively, are known to synthesize proteins of 19,000 Mr (19K) and 11.6K, but information regarding the identity and characterization of other potential E3 proteins encoded by the six remaining open reading frames (ORFs) is lacking. In this study, we show that the last ORF of region E3, which encodes a 14.7K protein, is expressed in adenovirus-infected cells. This information was largely derived from analysis of an E3 deletion mutant (H2dl801) in which an extensive deletion (1,939 base pairs) was found to eliminate all ORFs except for two proteins of 12.5K and 14.7K. The 14.7K protein was translated from RNA isolated from H2dl801-infected cells that had been hybridization selected to E3 DNA; hybridization-selected RNA from wild-type adenovirus type 5-infected cells translated both the 19K and the 14.7K proteins. Moreover, an antiserum directed against a bacterial 14.7K fusion protein (A. E. Tollefson and W. S. M. Wold, J. Virol. 62:33-39, 1988) immunoprecipitated the 14.7K translation product synthesized by wild-type and mutant H2dl801 adenovirus mRNAs.

The early region 1A (E1A) gene of adenovirus 5 encodes two proteins, 289AA and 243AA, which are translated from mRNAs of 13S and 12S, respectively. These two E1A proteins are identical except for an internal stretch of 46 amino acids unique to the larger protein. The 289AA protein activates transcription from promoters of other early adenoviral genes. The adenovirus type 5 host range mutants hr3, hr4, and hr5 are unable to activate transcription of these early viral genes. We show here that hr3, hr4, and hr5 each contain a distinct missense mutation in the E1A gene. We first localized the mutations in a series of constructed wild-type-hr hybrid E1A genes by using a biological assay which can discriminate between functional and nonfunctional E1A proteins. We then identified the mutations by DNA sequencing. In hr3 lysine replaced methionine at position 176, and in hr4 phenylalanine replaced leucine at position 173; both substitutions occurred in the region unique to the 289AA protein. In hr5, due to the splicing patterns of the two mRNAs, asparagine replaced serine as the last amino acid in the unique region of the 289AA protein at position 185, while aspartic acid replaced glycine at position 139 in the 243AA protein, which is the last amino acid common to both proteins before the unique region. These results substantiate the role of the 289AA protein in transcriptional activation and underscore the importance of the unique region as the basis of the functional difference between the two E1A proteins. Implications as to how these mutations affect the structure and function of the E1A proteins in transcriptional activation and transformation are discussed.

The appearance and steady-state accumulation of specific viral RNAs during the early phase of adenovirus type 5 (Ad5) infection was examined. HeLa cells were synchronously infected and harvested at 30 min intervals throughout the first 12 h of infection. Total cytoplasmic RNA was extracted from infected cells and analyzed by hybridization-selection and translation to identify the viral mRNAs from each early region on the basis of the protein products they encode. The same RNA samples were used for S-1 nuclease and Northern blot analyses to quantitatively compare the levels of individual viral RNAs that accumulate within each early transcription region (E1A, E1B, L1, E2A, E3 and E4). The salient features of this analysis show that RNA accumulation occurs first from E1A followed by E2A, E3 and E4, E1B and lastly, L1. Although the profile of RNA accumulation was unique for each early region, overlapping RNAs within E1A, E3, and E4, respectively, remained generally parallel to one another throughout early infection, in contrast to RNAs from E1B and L1, respectively. Since both the appearance and quantitative accumulation of specific early viral mRNAs were examined at many time points, a number of subtleties associated with the complex dynamics of early Ad5 gene expression were revealed. In particular, the L1 region was shown to transcribe from the major late promoter two early RNAs of 3.81 Kb and 3.5 Kb, either or both of which encode the 52,55 kDa proteins; the auxiliary i leader sequence was found on the 3.81 Kb RNA but not on the 3.5 Kb RNA.

We examined the ability of influenza-specific cytotoxic T lymphocytes to lyse adenovirus-transformed cells infected with influenza virus. Cytotoxic T lymphocyte lysis of Ad12-transformed cells was greatly reduced relative to that of Ad5-transformed cells. Lysability of adenovirus-12-transformed cells was restored in parallel with interferon-gamma induced increases in major histocompatibility complex class I gene products. These findings establish that recognition of foreign molecules by self-restricted cytotoxic T lymphocytes is reduced in adenovirus-12-transformed cells. This provides further evidence that Ad12 tumorigenicity is related to its ability to suppress class I major histocompatability complex molecule expression thereby avoiding recognition by the host immune system.

The E1A gene of adenovirus type 5 encodes two major proteins of 289 and 243 amino acid residues, which are identical except that the larger protein has an internal stretch of 46 amino acids required for efficient trans-activation of early viral promoters. This domain contains a consensus zinc finger motif (Cys-Xaa2-Cys-Xaa13-Cys-Xaa2-Cys) in which the cysteine residues serve as postulated ligands. Atomic absorption spectrophotometry applied to bacterially expressed E1A proteins revealed that the 289-amino acid protein binds one zinc ion, whereas the 243-amino acid protein binds no zinc. Replacing individual cysteine residues of the finger with other amino acids destroyed the trans-activating ability of the 289-amino acid protein, even when structurally or functionally conserved amino acids were substituted. These results strongly suggest that the zinc finger of the 46-amino acid domain is intimately linked to the ability of the large E1A protein to stimulate transcription of E1A-inducible promoters. Furthermore, zinc binding to one of the mutant finger proteins suggests either that only a precise finger structure formed by the tetrahedral coordination of zinc to the four consensus ligands is required for trans-activation or, possibly, that one of several neighboring histidine residues in various combinations with three of the consensus cysteine residues normally coordinates zinc. How the zinc finger in E1A might interact with DNA or protein to bring about trans-activation is discussed.

Adenovirus E1A and c-myc genes are known to be capable of transforming primary rat cells when they occur in combination with either polyoma middle-T or T24 Harvey-ras1 genes. There was a low level of amino acid sequence homology between the nuclear adenovirus-12 (Ad12)E1A protein product (289 amino acids) and the c-myc protein based on optimal alignment and percentage identity. In contrast to others [Ralston R, Bishop JM (1983) Nature 306:803-806], we concluded that this low level of amino acid sequence homology was not significant, since rabies glycoprotein (RGP), which has no transforming function and localizes to the cell surface, had a similar low level of amino acid sequence homology to the c-myc protein. Furthermore, dot-matrix analysis, when used to test the overall level of amino acid sequence homology, showed no significant homology between c-myc and Ad12E1A, E1B, or RGP. Thus, low levels of amino acid sequence homology between two proteins may not be sufficient to predict structural and functional similarities between them reliably, even if the two proteins appear to share a common function.

Infection of normal human melanocyte and nevus cultures with an adenovirus 12-Simian Virus 40 hybrid virus (Ad12-SV40) produced transformed cells that expressed SV40-T antigen. The Ad12-SV40 cells exhibited rapid cell proliferation to high cell densities and efficient growth in soft agar, but none of 15 transformed melanocyte and nevus cultures formed tumors when injected s.c. or under the renal capsule into athymic nude mice. While the Ad12-SV40-transformed cells lost certain properties associated with the melanocytic phenotype, i.e., pigmentation, tyrosinase activity and melanosome content, the expression of melanoma-associated antigens, including nerve growth factor receptor, p97 melano-transferrin, and chondroitin sulfate proteoglycan, remained stable. The transformed melanocytes acquired the ability to express HLA-DR antigen, which is found on nevus and melanoma cells. Total ganglioside patterns in Ad12-SV40-transformed cells changed to reflect more advanced stages of tumor progression. Transformed melanocytes, like nevus and melanoma cells, showed increased GD3 content and transformed nevus cells increased GD2 which is a feature of malignant melanoma cells. Ad12-SV40-transformed human melanocytes and nevus cells are useful tools for studying tumor progression under experimental conditions.

Antisera were prepared against the amino acid sequences encoded within the N-terminal half of the adenovirus 12 (Ad12) early region 1A (E1A) gene. This was accomplished by construction of a plasmid vector which encoded the N-terminal 131 amino acids of Ad12 E1A joined in frame to the coding sequence of beta-galactosidase. After induced synthesis in Escherichia coli, the Ad12 E1A-beta-galactosidase fusion protein (12-1A-FP) was extracted with urea and used to raise antibodies in rabbits. The 12-1A-FP antisera immunoprecipitated major phosphoproteins of 39,000 and 37,000 apparent molecular weights from Ad12-transformed and infected cells. The 12-1A-FP antisera also immunoprecipitated E1A phosphoproteins from Ad5-transformed and infected cells. Immunospecificity of the 12-1A-FP antisera was demonstrated by the ability of 12-1A-FP antigen to block immunoprecipitation of E1A proteins. Furthermore, E1A proteins immunoprecipitated from in vivo-labeled cells comigrated with those translated in vitro by RNA that had been hybridization selected to E1A DNA.

The adenovirus type 12 (Ad12) early region 1A (E1A) gene is thought to play a major role in repressing class I major histocompatibility complex expression in transformed rodent cells. However, since transformation by adenovirus requires both E1A and E1B genes, it has not been demonstrated whether the Ad12 E1A gene acts alone or synergistically with the E1B gene to accomplish this effect. Moreover, it is not known whether the repression of class I antigen synthesis by Ad12-transforming gene products occurs only in rodent cells. We show that the Ad12 E1A gene, in the absence of the E1B gene, is capable of greatly reducing the levels of class I HLA antigens and mRNAs in primary human cells transformed by the E1A gene of Ad12 and the large tumor antigen (T-antigen) gene of BK virus; control cells transformed by BK virus T-antigen gene alone or the highly related simian virus 40 T-antigen gene showed no apparent alteration in class I HLA expression. Human recombinant interferon gamma was able to restore synthesis of class I HLA antigens in transformed cells that produced Ad12 E1A proteins, indicating that these cells were not deficient for class I genes. These results strongly indicate that the Ad12 E1A proteins modulate class I gene expression by similar mechanisms in both transformed rodent and human cells.