Limited temperature-sensitive transactivation by mutant adenovirus type 2 E1a proteins.

A series of linker-scanning and deletion mutations was generated in the transactivating domain of the larger, 289-amino-acid-residue E1a protein of adenovirus type 2. Mutant genes were recombined into virus to assay the ability of the variant E1a proteins to activate expression of an E1a-dependent viral gene during infection. Results of assays performed at 32, 37, and 40 degrees C indicated that at least 2 of the 10 mutants tested showed limited temperature sensitivity for transactivation.     

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.     

Constructing chimeric type 12/type 5 adenovirus E1A genes and using them to identify an oncogenic determinant of adenovirus type 12.

The E1A gene of highly oncogenic type 12 adenovirus (Ad12) possesses a segment unique to this serotype and comprising 60 base pairs contiguous with and separating conserved regions 2 and 3 in the gene. A similar but slightly longer segment is also present in the E1A gene of highly oncogenic simian adenovirus type 7 (D. Kimelman, J. S. Miller, D. Porter, and B. E. Roberts, J. Virol. 53:399-409, 1985). This segment is missing entirely from the E1A gene of type 5 adenovirus, which is nononcogenic. To test the hypothesis that this unique separating or "spacer" region influences the oncogenicity of Ad12, we constructed ClaI and SmaI restriction sites on either side of it, which allowed reciprocal exchange between this and the equivalent cassette from type 5 adenovirus E1A, bounded by the same restriction sites intrinsic to that gene. The resultant Ad12-based chimeric viruses, ch702 and ch704, in which the spacer region is replaced with (in-frame) type 5 sequence, grow normally on human A549 cells and display wild-type transformation frequencies on baby rat and mouse kidney cells. In contrast, the oncogenic capacity of these chimeric viruses, as measured by tumor induction following virus inoculation in Hooded Lister rats, is greatly reduced. Likewise, cells transformed by ch702 and ch704 display reduced tumorigenicity compared with wild-type transformants in syngeneic rats. These results, coupled with recent preliminary tests using a mutant with a point mutation in this region, support the view that the unique spacer region of type 12 is an oncogenic determinant of this virus.     

Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215

The tumor suppressor p53 is important in the decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 posttranslational modifications and association with other proteins have been implicated in the regulation of its stability and transactivation activity. Here we show that p53 is phosphorylated by the mitotic kinase Aurora-A at serine 215. Unlike most identified phosphorylation sites of p53 that positively associate with p53 function (Brooks, C. L., and Gu, W. (2003) Curr. Opin. Cell Biol. 15, 164-171), the phosphorylation of p53 by Aurora-A at Ser-215 abrogates p53 DNA binding and transactivation activity. Downstream target genes of p53, such as p21Cip/WAF1 and PTEN, were inhibited by Aurora-A in a Ser-215 phosphorylation-dependent manner (i.e. phosphomimic p53-S215D lost and non-phosphorylatable p53-S215A retained normal p53 function). As a result, Aurora-A overrides the apoptosis and cell cycle arrest induced by cisplatin and gamma-irradiation, respectively. However, the effect of Aurora-A on p53 DNA binding and transactivation activity was not affected by phosphorylation of Ser-315, a recently identified Aurora-A phosphorylation site of p53 (Katayama, H., Sasai, K., Kawai, H., Yuan, Z. M., Bondaruk, J., Suzuki, F., Fujii, S., Arlinghaus, R. B., Czerniak, B. A., and Sen, S. (2004) Nat. Genet. 36, 55-62). Our data indicate that phosphorylation of p53 at Ser-215 by Aurora-A is a major mechanism to inactivate p53 and can provide a molecular insight for Aurora-A function.     

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.