Characterization of the 55K adenovirus type 5 E1B product and related proteins

In addition to major proteins of 19K and 55K (176 and 496 residues, 176R and 496R, respectively), early region 1B (E1B) of human adenovirus type 5 (Ad5) is predicted to encode at least three other polypeptides of 156R, 93R and 84R that share 79 amino-terminal residues with 496R. We have used a series of specific antipeptide sera to identify and partially characterize these proteins. 84R was produced in large amounts, 156R somewhat less, and 93R at very low levels. Synthesis of 176R, 496R, as well as the E2A 72K DNA-binding protein commenced shortly after that of E1A proteins in Ad5-infected KB cells. Production of 156R, 93R and 84R began somewhat later, but prior to the synthesis of the late structural protein IX and hexon. 156R, which is composed of the 79 amino-terminal and 77 carboxy-terminal amino acids of 496R, migrated on SDS-PAGE as two species which appeared to differ by their degree of phosphorylation. 156R and 496R yielded identical tryptic phosphopeptides that contained both phosphoserine and phosphothreonine, and one of these was immunoprecipitated by a serum specific for the carboxy terminus. These results suggested that Ser-490 and/or Ser-491 as well as Thr-495 are major sites of phosphorylation in these proteins.