Transformation-defective mutant of adenovirus type 5 containing a single altered E1a mRNA species

198

179

To determine whether the transcription regulatory activities of the adenoviral E1a gene play a role in its ability to transform primary cells we have constructed an extensive series of mutations within the E1a gene. The mutants have been characterized for their ability to transactivate the adenoviral early promoters, repress the transcriptional stimulation of the polyoma virus enhancer, establish primary baby rat kidney cells in culture and cooperate with the activated Ha‐ras oncogene in morphologically transforming these cells. The mutant phenotypes reveal that: (i) the two transcription regulatory activities of E1a are separable since essential protein domains map within different regions of the protein; (ii) transactivation is unlikely to contribute significantly to E1a‐mediated transformation since several isolated mutants lost the ability to transactivate but were nevertheless efficient at transformation; and (iii) both establishment and oncogene cooperation are linked to enhancer repression suggesting that E1a transforms cells by the repression of a cellular enhancer.

Section: Discussionmentioning

confidence: 99%

Mutational analysis of the adenovirus E1a gene: the role of transcriptional regulation in transformation.

Schneider¹,

Fisher²,

Goding³

et al. 1987

198

179

“…It is known that early region 1A of adenovirus encodes multifunctional products. Studies utilizing adenovirus type 2 (Ad2) and 5 (AdS) mutated in this region have indicated that these proteins are required for transformation of rat embryo cells, establishment of primary cells in culture, replication of viral DNA and for the expression of the other early regions (Harrison et al, 1977;Shenk, 1979a, 1979b;Berk et al, 1979;Houweling et al, 1980;Carlock and Jones, 1981;Nevins, 1981; Montell et al, 1982;Rossini, 1983;Ruley, 1983). Two mRNAs (12S and 13S), resulting from differential splicing during RNA processing, are transcribed from early region IA at early times during infection (Berk and Sharp, 1978;Chow et al, 1979;Kitchingman and Westphal, 1980).…”

Section: Introductionmentioning

confidence: 99%

Two functions encoded by adenovirus early region 1A are responsible for the activation and repression of the DNA-binding protein gene.

Guilfoyle¹,

Osheroff²,

Rossini³

1985

Human adenovirus early region 1A (E1A) gene products differentially regulate the expression of early region 2A (E2A) encoding the DNA‐binding protein (DBP). In a microinjection system, plasmids containing the DBP gene associated with both its early (map coordinate 75) and late (coordinate 72) promoters, or only with the early promoter, are inefficiently expressed, and the presence of E1A DNA is required for full expression. In contrast, the E2A plasmid in which the DBP gene is associated solely with its late promoter, efficiently produces DBP, the synthesis of which is significantly inhibited by an E1A gene product. To identify which of the E1A products is responsible for either activation or repression of DBP gene expression, two E1A mutants (Ad5hr1 and Ad2/5pm975) have been tested in the microinjection system in the presence of different DBP plasmids containing either one or both promoters. The results obtained indicate that the product encoded by the E1A 13S mRNA is responsible for the stimulation of DBP produced from the early promoter and that the 12S mRNA codes for the product which represses the synthesis of DBP from the late promoter. These results were confirmed using clones in which the E2A early or late promoter was associated to the chloramphenicol acetyltransferase (CAT) gene and assayed for CAT activity after cell transfection in the absence or in the presence of wild‐type or mutant E1A plasmids, and we have also shown that this promoter‐dependent regulation is reflected in the relative amount of specific DBP mRNA.

“…Two closely related viral proteins encoded in E1A are expressed during the early phase of an adenovirus infection (Perricaudet et al, 1979). Only the larger of these two EIA proteins, which is 289 amino acids in length, has transcriptioninducing activity (Carlock and Jones, 1981;Ricciardi et al, 1981;Montell et al, 1982Montell et al, , 1984. The Ad5 mutant hr I (Harrison et al, 1977) is defective in this 289 amino acid ElA protein specifically (Ricciardi et al, 1981).…”

Section: Resultsmentioning

confidence: 99%

“…Of the two closely related EIA proteins ( 1979), only the larger is required for stimulating transcription from early viral promoters during infection of HeLa cells (Carlock and Jones, 1981;Ricciardi et al, 1981;Montell et al, 1982). Similarly, only the larger ElA protein is required for stimulating transcription of a non-viral gene newly introduced into cells (Gaynor et al, 1984;Montell et al, 1984).…”

Section: Resultsmentioning

confidence: 99%

Adenovirus E1A protein activation of an integrated viral gene.

Curtois¹,

Berk²

1984

Adenovirus early region 1A (E1A) protein stimulates transcription from five viral promoters during the early phase of infection. This protein also stimulates transcription from non‐viral genes which are newly introduced into cells by infection or transfection, but not from the endogenous copies of these non‐viral genes. Here we show that E1A protein induces expression of an early adenovirus gene integrated into the chromosomal DNA of stably transformed cells. This induction requires the continuous expression of the same E1A protein which stimulates transcription of newly introduced genes. Thus, although the activity of E1A protein is not highly sequence specific, since it can stimulate transcription from newly introduced non‐viral genes, the results reported here indicate that it displays some degree of sequence specificity in that an endogenous adenovirus transcription unit responds differently from most cellular genes.