The human adenovirus 2 (Ad2) transforming region is located in the left 11.1% of the viral genome and encodes two early transcription units, ElA and E1B. Based on the amino acid sequence deduced from the Ad2 E1B DNA sequence (Gingeras et al., J. Biol. Chem. 257:13475-13491, 1982), we have prepared antibodies against synthetic peptides, 8 to 16 amino acids in length, encoded at the NH2 and COOH termini of the major ElB-19K and ElB-53K tumor antigens. The antipeptide antibodies immunoprecipitated the targeted ElB-19K or ElB-53K tumor antigens from extracts of Ad2-infected cells. The specificity of the antipeptide antibody recognition of the EIB tumor antigens was confirmed by peptide competition studies. Antipeptide antibodies directed to the NH2 and COOH termini immunoprecipitated the ElB-19K and ElB-53K tumor antigens from two Ad2-transformed rat cell lines, F17 and F4, providing evidence that identical tumor antigens are synthesized in Ad2-infected and Ad2-transformed cells. These results show that the ElB-19K and ElB-53K T antigens are not processed proteolytically at either the NH2 or COOH terminus. Our data provide strong evidence at the protein level that the ElB-19K and ElB-53K tumor antigens partially overlap in DNA sequence, with the ElB-19K initiating translation at the first ATG at nucleotide 1711 in translation reading frame 1 and the ElB-53K tumor antigen initiating translation at the second ATG at nucleotide 2016 in reading frame 3. This confirms the results of others on the N-terminal amino acid sequence of ElB-19K and theoretical deductions based on the DNA sequence. Our findings prove that the large ElB-53K T antigen initiates translation at the second ATG at nucleotide 2016 and not at equally plausible initiation codons located farther downstream at nucleotides 2202 and 2235. Thus, the ElB-53K T antigen is another example of a protein which initiates translation at an internal ATG rather than at the 5'-proximal ATG. During the early stages of adenovirus 2 (Ad2) productive infection, seven regions of the viral genome are transcribed, early region 1A (ElA), E1B, E2A, E2B, E3, E4, and late region 1 (Li) (see reference 45 for a recent review). EIA, located at map position 1.3 to 4.5, and E1B, located at map position 4.6 to 11.1, are of special interest because they encode the information for Ad2-induced cell transformation. Much is known about El of Ad2 and the closely related AdS (both group C). The DNA encoding the transforming genes has been sequenced (7, 16, 57). The major El-coded mRNAs and proteins have been identified by cell-free translation and by immunoprecipitation with antisera against Ad-transformed cells (Ad tumor sera). Cell-free translation studies with Ad2 and AdS El mRNAs have detected about eight proteins that are encoded by ElA and E1B (7, 13, 17, 23, 24, 35, 37, 38). As determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, two early proteins of apparent Mr 41,000 to 53,000 are translated from the ElA-13S mRNA, two early proteins of apparent Mr 35,000 to 4...

The adenovirus ElA transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Adl2) 13S and 12S mRNAs encode ElA T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Adl2 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the ElA T antigens synthesized in Adl2-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000,