Early E1B Research Articles

Readthrough activation of early adenovirus E1b gene transcription.

In cells productively infected with adenovirus type 5, transcription is not terminated between the E1a gene and the adjacent downstream E1b gene. Insertion of the mouse beta(maj)-globin transcription termination sequence (GGT) into the E1a coding region dramatically reduces early, but not late, E1b expression (E. Falck-Pedersen, J. Logan, T. Shenk, and J. E. Darnell, Jr., Cell 40:897-905, 1985). In the study described herein, we showed that base substitution mutations in the globin DNA that specifically relieved transcription termination also restored early E1b promoter activity in cis, establishing that maximal early E1b expression requires readthrough transcription originating from the adjacent upstream gene. To identify potential targets of readthrough activation, a series of recombinant viruses with double mutations was constructed. Each double-mutant virus strain had the transcription termination sequences in the first exon of E1a and a deletion within the transcription control region of E1b. Early E1b expression from the double-mutant strains was more defective than that from strains containing either mutation alone, indicating that the deleted regions (positions -362 to -35) are not the target for readthrough activation. Two findings suggested that a cis-dominant property of early viral templates is important for readthrough activation. First, the early E1b defect caused by the GGT insertion was not complemented in trans by factors present in late-infected cells. Second, restoration of E1b transcription at late times occurred concurrently with viral DNA replication. Readthrough activation may help convert virion DNA into a transcriptionally competent template prior to DNA replication and late transcription.

Antibodies to E1b Protein-Derived Peptides:of Enteric Adenovirus Type 40 Are Associated with Celiac Disease and Dermatitis Herpetiformis

Sera from 41 children with untreated celiac disease (CD), and 16 children with dermatitis herpetiformis (DH), and 57 matched controls were studied for serum antibodies to synthetic peptides derived from an early E1b protein of adenovirus type 12 and type 40 (Ad12, Ad40) and A-gliadin. In addition to peptides which share homology with A-gliadin, "nonhomologous" peptides derived from Ad12 and Ad40 E1b proteins were used as antigens in ELISA. Patients with CD and DH had significantly higher IgG antibody levels than those of controls to the nonhomologous Ad40 E1b peptide. Concomitant presence of antipeptide IgA antibodies to A-gliadin and Ad12 E1b or Ad40 E1b peptides (which share no homology with A-gliadin) increased the risk of CD/DH suggesting that both adenovirus infections and gluten exposure contribute to the development of CD/DH.

A Multicomponent cis-Activator of Transcription of the E1b Gene of Adenovirus Type 5

We report that transcription of the adenovirus type 5 E1b gene is activated substantially in cis by sequences located between positions -362 and -49 with respect to the RNA start site. DNA fragments consisting of the -362 to -49 sequences, or subsets thereof, were inserted into a reporter plasmid containing a minimal E1b promoter (positions -48 to +14) joined to the Escherichia coli cat gene. In the presence of cotransfected E1a and E1b genes in trans, CAT enzyme synthesis in transfected KB cells was stimulated about 20-fold by sequences from -362 to-49 (XY) in cis and to a lesser extent by sequences from either -362 to -128 (X) or -127 to -49 (Y). Adenoviruses were isolated lacking the X, Y, or XY sequences and KB cells were infected with one of the mutants, as well as wild-type virus to provide E1a and E1b in trans. Deletion of both X and Y resulted in a 20-fold reduction in early E1b RNA and a 12-fold reduction in late RNA. Deletion of X or Y alone produced up to 5-fold reductions in early or late E1b RNA accumulation. In vitro DNA-protein interactions in the Y sequence were revealed by modification of the procedure used for previous detection of X region footprints. These data indicate that X and Y sequences, which include E1a protein coding and 3′ untranslated DNA, also participate in DNA-protein interactions necessary for high levels of E1b promoter activity. The presence of such overlapping genetic elements raises the interesting possibility that functional E1a and E1b mRNAs must be synthesized from separate templates.

Antipeptide Antibodies to Adenovirus E1b Protein Indicate Enhanced Risk of Celiac Disease and Dermatitis herpetiformis

The relationship between adenovirus type 12 (Ad12), celiac disease (CD) and dermatitis herpetiformis (DH) was evaluated by enzyme-linked immunosorbent assay (ELISA). Diagnostic phase serum samples from 44 children with CD, 16 children with DH and 60 matched controls were studied for serum antibodies to synthetic peptides derived from an early E1b protein of Ad12 and A gliadin. Both the patient groups had significantly (p < 0.001) higher IgG antibody levels to the Ad12 E1b peptide than the controls. The difference was especially pronounced for girls (p < 0.005). Antipeptide IgG antibodies to Ad12 E1b and A gliadin posed a synergistic increase in the CD and DH risk suggesting that infection with Ad12 is associated with CD and DH.

Interleukin 6 enhances a cellular activity that functionally substitutes for E1A protein in transactivation.

An interleukin 6 (IL-6)-regulated cellular activity in HepG2 cells is found to functionally substitute for the transcriptional transactivator product of the adenovirus transforming gene E1A in transactivating E1A-dependent and E1A-responsive viral early genes. Mutant viruses deficient in E1A expression replicate in HepG2 cells. Induction with IL-6 leads to significant enhancement of synthesis of viral early E1B and E2ae mRNAs by greater than 30-fold and increases viral replication to the wild-type levels. The E1A-substituting activity activates E1A-responsive promoters in transient transfection, and this transcriptional activity is regulated by IL-6 induction. Formation of distinct protein-promoter complexes by binding of proteins in nuclear extracts prepared from HepG2 cells to the E1A-dependent E2ae promoter further supports the possibility that this activity may be a nuclear component in the IL-6 signal transduction pathway.

Cis-dominant defect in activation of adenovirus type 5 E1b early RNA synthesis

A cis-dominant mutation in the adjacent E1a gene disrupted the accumulation of adenovirus type 5 E1b mRNA during the early phase of infection. Steady-state levels of cytoplasmic and nuclear E1b RNAs in cells infected with dl312, a strain that lacks the E1a TATA box, cap site, and much of the coding sequence, were reduced 5- to 10-fold even when the E1a activator was provided in trans. The strain was defective for early E1b RNA synthesis but not for E1b RNA made late or during prolonged incubation in the presence of an inhibitor of DNA replication. The defect in E1b RNA synthesis could not be attributed to the E1a promoter sequences missing in dl312 DNA. If the E1a protein-coding region contains cis-acting regulatory sequences, they are not part of the previously mapped E1b transcriptional control region and may represent additional regulatory elements that ensure prompt and efficient E1b expression during the early phase of infection.

Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses.

Abstract We have studied the proteins encoded by the transforming region of the closely related human adenovirus serotypes 2 and 5. Messenger RNAs complementary to the two parts of this region, E1A and E1B, were prepared separately by hybridization to cloned DNA fragments encompassing 0.8 to 4.5 map units (for E1A) and 9.8 to 11.1 map units (for E1B). These RNAs were further fractionated by electrophoresis through agarose gels containing methylmercuric hydroxide, and then translated in vitro to identify the proteins encoded by each RNA species. E1A and E1B RNAs isolated at early and at late times after infection were compared. Three size classes of E1A mRNA direct the synthesis of at least five proteins: a28K ‡ protein encoded by a 0.6 kb mRNA, 42K and 54K proteins encoded by a 0.9 kb mRNA(s), and 48K and 58K proteins encoded by a 1.1 kb mRNA(s). The mRNA for the 28K protein accumulates preferentially at late times. Three size classes of early E1B mRNA direct the synthesis of three proteins: a 15K protein encoded by a 0.9 kb mRNA, an 18K protein encoded by a 1.2 kb mRNA, and a 57K protein encoded by a 2.6 kb mRNA. The mRNA for the 15K protein continues to accumulate at late times, and an additional 22K protein is made, while the 18K and 57K proteins are synthesized poorly, if at all, with late RNA. Substantially different E1A and E1B proteins are encoded by RNA from cells infected with the adenovirus type 5 mutants dl311, dl312, dl313, dl314 and hr1, which are all defective for replication on human cells and, except for dl311, for transformation. dl312, dl314 and hr1 are also defective for early viral gene expression. No viral mRNA could be detected in either dl312 or dl314-infected cells. hr1-infected cells contain a 0.9 kb mRNA encoding E1A 54K and 42K, but instead of 58K and 48K, the 1.1 kb hr1-E1A mRNA is translated into a 26K protein. The E1B mRNAs are present in substantially decreased amounts in hr1-infected cells. dl311-infected cells contain E1A mRNAs of 1.1 and 0.9 kb, encoding 38K and 34K proteins, respectively, and normal E1B mRNAs. The dl313 mRNAs of 1.1 and 0.9 kb contained fused E1A and E1B sequences and were translated into 40K and 36K proteins, respectively. These results are related to the mRNA structures and the biological activity of regions of the individual proteins.