Adenovirus E1B 55-kilodalton oncoprotein inhibits p53 acetylation by PCAF.

The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcriptional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation of p53 by these proteins enhances p53 sequence-specific DNA binding. Here we show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF in vivo and in vitro, while acetylation of histones and PCAF autoacetylation is not affected. Furthermore, the DNA-binding activity of p53 is diminished in cells expressing the E1B 55-kDa protein. PCAF binds to the E1B 55-kDa protein and to a region near the C terminus of p53 encompassing Lys-320, the specific PCAF acetylation site. We further show that the E1B 55-kDa protein interferes with the physical interaction between PCAF and p53, suggesting that the E1B 55-kDa protein inhibits PCAF acetylase function on p53 by preventing enzyme-substrate interaction. These results underscore the importance of p53 acetylation for its function and suggest that inhibition of p53 acetylation by viral oncoproteins prevent its activation, thereby contributing to viral transformation.

Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.

The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E. White, Genes Dev. 10:1-15, 1996). To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J. Han, P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White, Genes Dev. 10:461-477, 1996), and another is Bak (S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J.-C. Martinou, and R. Brown, Nature (London) 374:731-733, 1995). Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis. Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K. Nbk contained BH3 but not BH1 or BH2. It also interacted with Bcl-2 but not with Bax. Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific. In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes. Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K. Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis. Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein.

The quaternary structure of the adenovirus 12 early region 1B 54K protein.

The quaternary structure of the adenovirus early region 1B 54K protein has been examined under denaturing and nondenaturing conditions. In the presence of SDS the protein has a strong tendency to form disulfide-linked high-molecular-weight polymers. Under nondenaturing, but reducing, conditions the in vitro-translated 54K polypeptide forms oligomers (probably tetramers) of molecular weight approximately 2 x 10(5). After subcellular fractionation of Ad12 early region 1-transformed cells, the 54K E1B protein present in the cytoplasm had a molecular weight similar to that determined for the in vitro-translated material. However, two populations of the viral protein could be distinguished in the nucleus-one of a size similar to that seen in the cytoplasm and the other of appreciably higher molecular weight (approximately 2 x 10(6)). No difference in migration pattern was observed after treatment of the nuclear extract with DNase I or RNase. A proportion of the Ad12 E1B 54K protein in both the high- and the low-molecular-weight populations in the nucleus was found to form a complex with p53, and it is therefore concluded that the very high molecular weight derives from interaction with an, as yet, unidentified component.

Enteric adenovirus type 40: expression of E1B proteins in vitro and in vivo.

The genes encoding the enteric adenovirus type 40 E1B proteins designated 19K, 55K, and 15K (55K related) have been cloned into the pET3a expression vector and synthesized by in vitro transcription and translation and by in vivo expression after induction in bacteria. The 19K product expressed in bacteria is recognized by anti-peptide sera specific for the C-terminal region of the open reading frame and has the same M(r) as 19K protein immunoprecipitated from virus-infected cells. The 55K protein synthesized in bacteria is insoluble except under extreme denaturing conditions, but after in vitro transcription followed by translation, a polypeptide of the predicted size is obtained. The 15K protein, equivalent to the first 73 and last 29 of the 476-residue 55K protein with an internal deletion of 374 amino acids, is expressed to a high level in bacteria in a soluble form and interacts weakly but specifically with N- and C-terminal anti-peptide sera. The bacterially expressed 15K protein was used to raise antibodies in rabbits. This serum precipitates the 55K protein expressed by in vitro translation, but only the 15K product can be immunoprecipitated from virus-infected cells. The same antiserum, however, detects the 55K protein in infected cells by Western blotting, at a time broadly coinciding with the onset of DNA replication. This is the first identification of Ad40 55K protein in infected cells and confirms that the Ad40 22S mRNA can be utilized in vivo. The question of whether this protein is functional can now be addressed.

Expression of the adenovirus E1B 175R protein and its association with membranes of Escherichia coli.

The E1B 175-amino-acid (175R) protein of adenovirus 2 is required for cellular transformation of primary cells and establishing cell morphology in lytically infected cells. To investigate the biochemical function of this protein, we constructed a bacterial expression vector (pKHB1-T) to produce the 175R protein in sufficient amounts for purification and biochemical analysis. On the basis of DNA sequencing, gel electrophoresis, and immunoblot analysis, the pKHB1-T-encoded 175R protein appears to be identical to that expressed transiently in mammalian or adenovirus-transformed cells. The bacterially produced viral protein was also found to be quite stable and without any modifications. Partial purification of the pKHB1-T-encoded protein revealed that the majority of its associates with the inner membrane of the bacterial cell. This, together with the possibility of the 175R protein containing an N-terminal amphipathic alpha-helix as a potential translocation signal, suggests that there may be a common mechanism of protein transport operating in both eucaryotic and procaryotic systems.