Cell-free translation of adenovirus 2 E1a- and E1b-specific mRNAs and evidence that E1a-related polypeptides are produced from E1a-E1b overlapping mRNA.
We have characterized the polypeptides translated in vitro by mRNAs of early region 1 (E1) of human adenovirus (Ad) type 2. Poly (A+) polyribosomal RNA was isolated from early Ad2-infected cells, the viral specific mRNAs were selected by hybridization to Ad2 E1a and E1b DNA, and the mRNAs were translated in vitro using [35S]methionine as a labeled precursor with a rabbit reticulocyte lysate. E1a-selected mRNA was translated to the 45-58-kDa cluster of polypeptides. We show here that E1b-selected mRNA can also be translated to the 45-58-kDa cluster of polypeptides in addition to the major 19-kDa polypeptide. The E1b 58-kDa polypeptide was produced only at a low level unless E1b mRNA is fractionated before translation to enrich for the 58-kDa mRNA. Translation of E1b region-selected mRNAs that have been fractionated by size shows that the 22 S mRNA fraction is translated to at least the 53-58-kDa E1a-related polypeptides as well as to E1b 58- and 19-kDa polypeptides. Our experiments suggest that the 22 S mRNA fraction includes E1a-E1b overlapping mRNA which was translated to E1a-related polypeptides as well as E1b 22 S mRNA. When compared by two-dimensional gel electrophoresis and by tryptic peptide mapping, the cluster of polypeptides translated from E1a-selected mRNA and the cluster translated from E1b-selected mRNA were distinguishable. A possible explanation for this is discussed, based upon splicing sites of the E1a-E1b overlapping mRNA which would result in an amino acid sequence with a COOH-terminal end slightly different from that of E1a polypeptides.
D000260:Adenoviruses, Human / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism; D005470:Fluorometry; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000032:analysis; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis*
D004279:DNA, Viral; D010446:Peptide Fragments; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D011061:Poly A; D012313:RNA; D014357:Trypsin