Functions of purified E1A protein microinjected into mammalian cells

Abstract

We have purified the human type C adenovirus E1A 13S mRNA gene product, expressed in Escherichia coli, and demonstrate that the protein exhibits genuine viral functions upon microinjection into mammalian cells. We show that the E1A protein activates expression of the adenovirus E2A gene and induces expression from the major late transcription unit of the adenovirus E1A deletion mutant, H5dl312. We use this functional assay to examine the stability of E1A protein microinjected into cells and find that E1A exhibits full function for at least 18 hours after its injection. In addition, the purified E1A protein was used to generate a high-titer monospecific rabbit antiserum. This antiserum was used to detect and localize E1A proteins within adenovirus-infected cells as well as within microinjected cells. The E1A protein is found to rapidly and quantitatively localize to the cell nucleus following microinjection into the cell cytoplasm. Thus, nuclear localization is an intrinsic property of the E1A polypeptide. The ability of the E1A protein to localize to the cell nucleus and to induce expression from the H5dl312 major late transcription unit is shown to be highly heat stable.