Mutations in CR1 of E1A 12S yield dominant negative suppressors of immortalization and the lytic cycle.

The Adenovirus 5 E1A 12S gene is responsible for the establishment of immortalization of primary cells by Adenovirus. We have identified two mutants of 12S (30K and NTdl814), which, when coexpressed with wild-type 12S in primary baby rat kidney cells, were capable of suppressing the immortalizing function of the wild-type 12S gene, even when the mutant proteins were expressed at levels lower than wild type. 30K and NTdl814 did not affect the ability of the coexpressed 12S to activate the cell cycle, but have a suppressive effect on 12S-induced DNA synthesis and proliferation at late times in the immortalization pathway. Both the dominant negative mutants have a deletion in conserved region (CR)1 in the first exon of E1A, which encompasses one of the pRb-family binding regions. However, the mutants did not effect the binding of cellular proteins to full-length 12S. A suppressive effect on wild-type 12S was not observed with mutants that have lost any other region or function. In addition, expression of 30K, which is equivalent to the protein encoded by the 10S mRNA of E1A, inhibited E1A function in lytic cycle. Thus, loss of the CR1 seems to be a prerequisite for a mutant to have a dominant negative effect on E1A functions.