Evidence that AGUAUAUGA and CCAAGAUGA initiate translation in the same mRNA region E3 of adenovirus.

We described a simple method to introduce site-specific mutations into region E3 of adenovirus (Ad). Mutations are made in cloned Ad2 EcoRI-D (map position 76-83), then ligated between Ad5 EcoRI-A (map position 0-76) and EcoRI-B (map position 83-100) to complete the viral genome. We have used this method to isolate a viable virus mutant (dl702) that is relevant to the problems of translation initiation and gene organization in the E3 complex transcription unit. mRNA a in region E3 encodes an abundant glycoprotein termed gp19K. There are two AUGs in mRNA a that are 5' to AUG1204 which initiates gp19K. One of these, AUG1022, could initiate a 6.7K protein, although this protein has not been identified in infected cells. Mutant dl702 has a deletion such that the 6.7K gene is fused in-frame to the gp19K gene. We report that the 6.7K-gp19K fusion protein is synthesized both in dl702-infected cells and after cell free translation of infected cell RNA. The quantity of fusion protein made is much less than that of wild type gp19K. The sequence context of AUG1022 for 6.7K is AGUAUAUGA, and that of AUG1204 for gp19K is CCAAGAUGA. The consensus sequence of eukaryotic initiation codons is CCPuCCAUGG, with the Pu at -3 being important (M. Kozak, Nucleic Acids Res. 12, 857-872, 1984). Our results suggest that (i) AUG1022 can initiate translation in vivo and therefore the 6.7K protein probably is made in infected cells, (ii) that mRNA a is a dicistronic mRNA encoding the 6.7K and gp19K proteins, and (iii) that the initiation codon for 6.7K may be much less efficient than that for gp19K. Thus, the E3 genes may be organized such that the relative abundance of the 6.7K and gp19K proteins is controlled by the efficiency of their initiation codons in the same mRNA.