Adenovirus type 12 early region 1A proteins repress class I HLA expression in transformed human cells

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.     

Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation.

The expression of the adenovirus (Ad) early coding region 1a (E1a) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earlier studies have shown that the 289-amino acid protein is essential for cell transformation. We have constructed an Ad type 2 (Ad2) deletion mutant (dl231) in which the intervening sequence for the 13S mRNA is precisely removed. Mutant dl231 is completely viable in human KB cells and produces normal amounts of 13S mRNA but much reduced amounts of a defective 12S mRNA. Mutant dl231 induces focal transformation of established rat embryo fibroblasts at a frequency one-fifth to one-half that of wild-type virus. However, the transformed cells are defective in their ability to form anchorage-independent colonies on semisolid medium. Therefore, our results demonstrate that the 243-amino acid protein is required for full transformation of rat embryo cells.     

Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters.

Recombinant adenoviruses carrying the hepatitis B virus surface antigen coding sequence in the adenovirus E3 region were constructed using DNA from either adenovirus type 5 or an adenovirus type 5 E3-region deletion mutant. Both of these recombinant adenoviruses replicated as efficiently as wild-type adenovirus in all human cells tested, including the human diploid cell strain WI-38. This indicates that insertion of the hepatitis B virus surface antigen gene into the E3 region does not significantly affect viral replication. Human cells infected with these recombinant adenoviruses secreted immunoreactive hepatitis B virus surface antigen. Since a practical small animal model for human adenoviruses was lacking, a hamster model was developed to evaluate the immunogenic potential of these recombinant adenoviruses. Upon intranasal inoculation, both wild-type adenovirus and the adenovirus E3-region deletion mutant replicated in the lungs of these animals and induced an antibody response against adenovirus. Hamsters similarly immunized with the live recombinant adenoviruses produced antibody against both adenovirus and hepatitis B virus surface antigen.     

Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5'' -CCGG- 3'' methylation.

The plasmid construct pSVO-CAT has been used to test adenovirus promoter activities in the unmethylated or methylated state. We have now observed that the E2A late promoter of adenovirus type 2 (Ad2) DNA also activated the chloramphenicol acetyltransferase (CAT) gene upon transfection of the pAd2E2A-CAT construct into mammalian cells, and it was inactivated by specific methylations of three 5' -CCGG- 3' sites. Similar results had been reported previously after microinjecting promoter-methylated constructs into oocytes of Xenopus laevis. Surprisingly, it was found that the pSVO-CAT construct, which lacked eukaryotic promoter sequences, was able to express the CAT gene upon transfection into human or hamster cells that harbored and constitutively expressed the E1 region of Ad2 or Ad5 DNA. In these cells, the expression of the pAd2E2A-CAT construct was enhanced, but it was only partly sensitive to DNA methylation, possibly because DNA methylation was counteracted directly or indirectly by E1 functions. The pSVO-CAT construct was also expressed in HeLa or BHK21 cells upon cotransfection with a plasmid carrying the HindIII-G fragment of Ad2 DNA that contained the E1A region and part of the E1B region. By mapping pSVO-CAT-specific RNAs, we could demonstrate that pSVO-CAT activity in Ad2- or Ad5-transformed cells was mediated by prokaryotic promoter-like sequences in the pBR322 section of the construct, and it presumably functioned via trans-activation mediated by the E1 region. This trans-activation of pSVO-CAT in adenovirus-transformed cells was partly insensitive to DNA methylation.     

Mapping of functional domains in adenovirus E1A proteins.

We have modified the E1A gene of human subgroup C adenovirus by introducing deletions in its coding sequence. Various truncated E1A proteins were expressed in Escherichia coli, purified, and microinjected via glass capillaries into Vero cells. We monitored their movement from the cell cytoplasm to the nucleus and their ability to induce expression of H5dl312, an adenovirus E1A deletion mutant. Our results show that the carboxyl terminus of E1A contains sequences essential for rapid and efficient nuclear localization. Essential information for efficient H5dl312 complementation is contained in an internal region, comprising sequences of both exons of the E1A gene. A first exon-encoded region, however, is sufficient to induce low levels of adenovirus gene expression. Information for nuclear localization and for H5dl312 complementation are therefore encoded by distinct domains of the E1A gene. In addition, we determined that the human c-myc product was unable to complement H5dl312.     

Growth factor(s) produced during infection with an adenovirus variant stimulates proliferation of nonestablished epithelial cells.

Infection of primary baby rat kidney cells with an adenovirus variant that encodes only the 12S gene of the E1A region, adenovirus type 5 (Ad5) 12S, results in the production of a growth factor that stimulates primary epithelial cells to proliferate. Increased epithelial cell DNA synthesis and proliferation is detectable between 24 and 36 hr after the addition of conditioned medium from Ad5 12S infected cells and not from cells infected with an E1A deletion mutant virus, Ad5 dl312. This mitogenic factor(s) is effective in the absence of serum and can override the inhibitory effect of serum on primary epithelial cells. Furthermore, there is a requirement for the continued presence of the growth factor(s) in the Ad5 12S conditioned medium to maintain epithelial cell proliferation, and the conditioned medium can maintain these cells in a proliferative state for at least 6 wk. The stimulatory activity in Ad5 12S conditioned medium is associated with large molecular weight complexes, from which it can be released by 4 M NaCl. Several characteristics of the growth factor(s) indicate that it is a unique mitogen for epithelial cells.     

Transgenic mouse model for human gastric carcinoma.

To understand the pathogenesis that may be induced by human adenovirus type 12 (Ad12), we have generated transgenic mice carrying the Ad12 early region 1 under control of the mouse mammary tumor virus long terminal repeat. Eleven of 11 male founder mice, but only 2 of 12 females, died between 3 to 4 mo of age. Death was associated with presence of tumors at or near the squamocolumnar junction of the stomach. Microscopically, these multifocal tumors appeared to arise from hyperplastic epithelium and showed features consistent with adenocarcinoma or adenosquamous carcinoma. High levels of expression of both the Ad12 E1A and E1B genes were seen in the tumor-bearing stomach. Various levels of expression were also detected in other tissues, although the stomach was the only organ with detectable pathology. These observations suggest an organ-specific action of the Ad12 early region 1 gene products. This transgenic mouse model provides an experimental system for studying the development of human carcinomas at sites of transition from squamous to columnar epithelium.     

Characterization of a replication-incompetent adenovirus type 5 mutant deleted for the preterminal protein gene

An adenovirus type 5 mutant deleted for the preterminal protein (pTP) gene was constructed using cell lines that express pTP. The pTP deletion mutant virus is incapable of replicating in the absence of complementation and does not express detectable levels of viral mRNAs that are expressed only after the onset of replication. Accumulation of early-region mRNAs, including that for E1A, exhibits a lag relative to that observed from the wild-type virus. However, E1A mRNA accumulation attains a steady-state level similar to the level of expression during the early phase of infection with the wild-type virus. In 293-pTP cells (human embryonic kidney cells that express pTP in addition to high levels of adenovirus E1A and E1B proteins), the pTP deletion mutant virus replicates efficiently and yields infectious titers within 5-fold of that of the wild-type virus. The deletion of 1.2 kb of pTP-encoding sequence increases the size of foreign DNA that can be introduced into the virus and, with an absolute block to replication, makes this virus an important tool for gene therapy.     

Expression of early adenovirus genes requires a viral encoded acidic polypeptide.

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.     

Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.

Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells.