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

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.     

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter.

The transformation-defective Vero cell host range mutant CS-1 of the highly oncogenic adenovirus type 12 (Ad12) (Ad12-CS-1) has a 69-bp deletion in the early region 1A (E1A) gene that removes the carboxy-terminal half of conserved region 2 and the amino-terminal half of the Ad12-specific so-called spacer that seems to play a pivotal role in the oncogenicity of the virus. Despite its deficiency in immortalizing and transforming primary rodent cells, we found that the E1A 13S protein of Ad12-CS-1 retains the ability to bind p105-RB, p107, and p130 in nuclear extract binding assays with glutathione S-transferase-E1A fusion proteins and Western blot analysis. Like wild-type E1A, the mutant protein was able to dissociate E2F from retinoblastoma-related protein-containing complexes, as judged from gel shift experiments with purified 12S and 13S proteins from transfection experiments with an E1A expression vector or from infection with the respective virus. Moreover, in transient expression assays, the 12S and 13S products of wild-type Ad12 and Ad12-CS-1 were shown to transactivate the Ad12 E1A promoter containing E2F-1 and E2F-5-motifs, respectively, in a comparable manner. The same results were obtained from transfection assays with the E2F motif-dependent E2 promoter of adenovirus type 5 or the human dihydrofolate reductase promoter. These data suggest that efficient infection by Ad12 and the correlated virus-induced reprogramming of the infected cells, including the induction of cell cycle-relevant mechanisms (e.g. E2F activation), can be uncoupled from the transformation properties of the virus.     

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.