The binding sites for the 300-, 107-, and 105-kilodalton cellular proteins which associate with human adenovirus type 5 ElA products were studied with ElA deletion mutants. AB appeared to bind to the amino-terminal half of ElA products in regions necessary for oncogenic transformation. These results suggest that these cellular species may be important for the biological activity of ElA products.

We previously purified and characterized functionally the Escherichia coli-expressed product of the human subgroup C adenovirus E1A 13S mRNA (B. Ferguson, N. Jones, J. Richter, and M. Rosenberg, Science 224:1343-1346, 1984; B. Krippl, B. Ferguson, M. Rosenberg, and H. Westphal, Proc. Natl. Acad. Sci. USA 81:6988-6992, 1984). We have now expressed in E. coli and purified the protein product encoded by the human subgroup C adenovirus E1A 12S mRNA and have compared the functional properties of this protein with those of the E1A 13S mRNA product. Using microinjection techniques to introduce these proteins into mammalian cells, we found that the E1A 12S mRNA product, like the 13S mRNA product, localized rapidly to the cell nucleus and induced adenovirus gene expression. Although both E1A gene products localized to the nucleus and stimulated adenovirus gene transcription, these proteins did not directly bind to DNA under conditions in which a known DNA-binding protein, the human c-myc gene product, bound DNA efficiently. Thus, the E1A and myc gene products, which have been related both structurally and functionally, exhibit distinctly different biochemical properties.

The adenovirus E1a gene products are nuclear proteins important in transcriptional control of viral functions during infection. By producing normal E1a proteins and derivatives of E1a in bacteria and microinjecting these proteins into cultured cells, we were able to examine their ability to localize to the nucleus. We showed that a short peptide sequence at the carboxyl terminus of E1a is necessary for the rapid (30-min) nuclear localization of that protein. Additionally, we showed that just the last five amino acids of E1a are sufficient to direct nuclear accumulation of a heterologous protein, Escherichia coli galactokinase, with the same kinetics as native E1a. The mechanism by which this pentamer mediates rapid nuclear localization was examined by testing the ability of a galactokinase derivative which has no signal pentamer to exit the nucleus, as well as to enter it. Because neither free entry nor exit was detected, the effect of the signal is unlikely to be through increased nuclear retention of freely diffusible proteins but rather by enhancement of entry into the nucleus.

The human type C adenovirus E1a 13S messenger RNA encodes a gene product, that positively regulates the transcription of viral genes and certain cellular genes and is involved in the transformation of primary mammalian cells. The E1a gene product was expressed at high levels in Escherichia coli. In a Xenopus oocyte microinjection assay, the purified Escherichia coli-produced protein activated the E1a-responsive adenovirus E3 promoter and functioned as efficiently as the E1a gene itself.