Transformation properties of type 5 adenovirus mutants that differentially express the E1A gene products.

Abstract

Type 5 adenovirus mutants that differentially express ElA 13S, 12S, or 9S mRNAs were constructed to study the role of their gene products in transformation. H5dl520 expresses the 243-amino-acid (AA) protein encoded in the 12S mRNA but not the 13S mRNA-encoded 289-AA protein. This mutant transformed both cloned rat embryo fibroblast (CREF) cells and baby rat kidney (BRK) cells at a frequency 40-100 times greater than did wild-type viruses. In addition, all of the foci produced were fibroblastic and grew very slowly at 320C. In contrast, H5dl21, which was mutated so that only the 54-AA protein encoded by the 9S mRNA was synthesized, did not transform either cell type. DNA transfection studies with plasmids from which these mutants were constructed demonstrated that the differences in transformation frequencies were not as marked. The plasmid pDl/D2, which directs the synthesis of the 54-AA protein only, was found to transform baby rat kidney cells at low frequency, provided the gene was linked to a fragment from the simian virus 40 genome containing the transcriptional enhancer element.The early region genes ElA and E1B, located at the lefthand end of the genome, have been shown to be essential for complete morphological transformation of rodent cells by human adenoviruses (Ad) (1-4). However, transfections of baby rat kidney (BRK) cells (5) or an established cloned rat embryo fibroblast (CREF) cell line (6) with various fragments of the Ad type 5 (AdS) or type 12 (Adl2) genome containing only the ElA sequences produce partial or incomplete morphological transformation, whereas DNA fragments containing only the E1B genes do not transform (7).The exact roles of the ElA genes in transformation are not well understood, but they do provide an establishment function that is concerned with the immortalization of primary cells (2, 8). The ElA sequences encode three different but related products. During early times of productive infection, the ElA primary transcript is differentially spliced to give 12S and 13S messages that differ in the extent of internal sequences removed by RNA splicing (9-12). The translational reading frames of both messages are identical, and, thus, the proteins encoded by each [a 289-amino-acid (AA) protein by the 13S mRNA and the 243-AA protein by the 12S mRNA] differ only by an additional 46 AA encoded by the sequences unique to the 13S mRNA. At late times of infection, a third message, 9S in size, is synthesized. The predicted polypeptide encoded by this message is 54 AA in size and has the same amino-terminal sequence before the splice as in the protein products of the 12S and 13S mRNAs (13). A wellcharacterized function of the ElA region in the productive infection of cells is to produce a product that hastens the transcription of the other early viral genes (4, [14][15][16] (3,5,18,20); some are transformation defective, whereas, others are cold sensitive for the initiation and maintenance of the transformed phenotype. From these results it was deduced that the 289-AA protein was di...

HPV E7 expression in skeletal muscle cells distinguishes initiation of the postmitotic state from its maintenance

“…The Ad-FLAG-E7 and Ad-wt E7 viruses were generated using the method devised by He et al (1998). The dl520 virus is a deletion mutant of human adenovirus type 5, expressing 12S but not 13S E1A (Haley et al, 1984). All recombinant adenoviruses except dl520, independent of the construction method, express their respective cDNAs under the control of the cytomegalovirus immediate-early promoter/enhancer.…”

Inhibition of p53-mediated transactivation and cell cycle arrest by E1A through its p300/CBP-interacting regionSelfcloseTable

“…The 243-amino acid 12S E1A protein contains the sequences necessary for rodent cell transformation, i.e. the transactivation function of E1A is not required (Haley et al, 1984). There are three regions in the 12S E1A protein which are required for transformation: (1) the nonconserved amino-terminus (aa 2 to 24), (2) the conserved region #1 (CR1; aa 40 to 80) and (3) the conserved region #2 (CR2; aa 120 to 140) (see Bayley and Mymryk, 1994 for review).…”

Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1.

“…ElA proteins also induce cellular division and immortalization as well as transformation of embryonic cells in conjunction with early region 1B (E1B) proteins (7,16,22,26,51,54). The regions of ElA responsible for these activities have been less clearly defined than those for transactivation and transcriptional inhibition, but at least two regions within exon 1 are required (34,35,49,52,63).…”