Domains required for in vitro association between the cellular p53 and the adenovirus 2 E1B 55K proteins

The 55K protein encoded by the adenovirus 2 E1B gene is required for complete cellular transformation and binds the cellular protein p53. Using an in vitro immunoprecipitation assay, we mapped the domains in both 55K and p53 required for the interaction of the two proteins. The domain in p53 mapped to the amino terminal 123 residues. There are several domains in the 495 residue 55K polypeptide which contribute to stable association with p53, with the most essential region mapping between residues 224 and 354. Mutations which prevented 55K-p53 binding were not more defective for transformation than other mutations which did not affect binding.     

An adenovirus early region 4 gene product is required for induction of the infection-specific form of cellular E2F activity

E2F is a cellular, sequence-specific DNA-binding factor that binds to pairs of sites that occur upstream of the E1A and E2 early mRNA cap sites. During adenovirus infection, there is induction of a form of E2F that binds cooperatively to the pair of sites in the E2 control region. Production of the infection-specific E2F activity is dependent on early region 4 (E4), as extracts of cells infected with a mutant that lacks E4 did not contain this activity. Instead, two new forms of E2F were seen with the E4 mutant. Infection with mutant viruses unable to make E1A gene products produced the wild-type infection-specific E2F activity after a delay. Mutations in the E1B-55 kD-, E1B-21 kD-, E2-72 kD-, and E3-coding regions had no effect on production of infection-specific E2F. Analysis of cell lines confirmed the results obtained with mutant viruses. Cells that expressed E1A but not E4 genes (e.g., 293 cells) did not contain infection-specific E2F. Cell lines that expressed the E4 gene contained the activity. These observations demonstrate that E4 participates in the infection-induced change in E2F-binding activity. The data are consistent with E1A playing an indirect role in the process by mediating the efficient expression of E4 gene products which, in turn, induce the alteration in E2F activity.     

The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex

Nuclear factor E2F is a cellular protein that binds to the adenovirus E2 promoter and E1A enhancer regions and to the cellular c-myc P2 promoter region. The DNA binding activity of E2F, detected in vitro using nuclear extracts prepared from HeLa cells, is increased by adenovirus infection (termed E2F induction). We demonstrate here that a 19.5-kD protein, encoded by adenovirus early region 4 (E4) open reading frame (ORF) 6/7, is primarily responsible for the induction of E2F DNA binding activity to the E2 promoter region. Viral mutants that contain frame-shift mutations in E4 ORF 6/7 failed to induce E2F binding activity; a virus that carries an E4 ORF 6/7 cDNA in place of the E4-coding sequences induced E2F efficiently. Using gel mobility shift assays, we demonstrate that the E4 ORF 6/7 product induces the binding of E2F to the E2 promoter via a direct complex. The addition of a peptide-specific antiserum, directed against the E4 ORF 6/7 protein, to an in vitro E2F-binding reaction resulted in the formation of a DNA-protein complex with reduced gel mobility compared to the normal, adenovirus-induced E2F-E2 promoter complex. The formation of the E2F-E2 promoter-antibody complex was blocked by the addition of the cognate peptide used to generate the antiserum but not by a nonspecific peptide. Nuclear extracts prepared from adenovirus-infected HeLa cells were cleared of E2F binding activity using the ORF 6/7 peptide-specific serum, but not the preimmune serum, suggesting that E2F and the E4 ORF 6/7 product form a protein-protein complex in solution. The adenovirus E1A proteins are not absolutely required for the induction of E2F binding activity because the infection of HeLa cells with an E1A mutant, dl312, at high multiplicity resulted in E2F induction. Under these conditions of infection, the E4 ORF 6/7 product was synthesized. E2F binding activity was induced, but inefficiently, in cells infected with E4 ORF 6/7 mutants, indicating that an additional pathway may lead to E2F induction.     

Phosphorylation of adenovirus E1A proteins by the p34cdc2 protein kinase.

Adenovirus early region 1A (E1A) products are phosphorylated nuclear oncoproteins which appear to derive transforming activity largely through interactions with cellular proteins including the tumor suppressor p105/Rb-1 and cyclin A (p60cycA), a regulatory subunit associated with p34cdc2 and the related protein kinase p33cdk2. We have identified several sites of phosphorylation on E1A proteins previously and showed that phosphorylation at Ser-89 alters electrophoretic mobility significantly and affects E1A-mediated transforming activity to some extent. We now report that both Ser-89 and Ser-219, the major E1A phosphorylation site, were phosphorylated in vitro by p34cdc2 purified from HeLa cells. We also found that E1A proteins seemed to be phosphorylated at the highest levels in vivo in mitotic cells which express maximal levels of p34cdc2 kinase activity. Thus, in addition to forming complexes with p60cycA, a regulator of p34cdc2 and related kinases, and p105/Rb-1 which exhibits cell cycle-dependent phosphorylation, E1A proteins seem to be substrates for p34cdc2. These data suggested that a link could exist between phosphorylation, cell cycle progression, and the regulation of transforming activity of E1A proteins.     

Herpes simplex virus induces intracellular redistribution of E2F4 and accumulation of E2F pocket protein complexes.

Accumulation of E2F-p107 and E2F-pRB DNA binding complexes occurred after herpes simplex virus infection of U2-OS cells. Accumulation of E2F-p107 also occurred by 4 h p.i. in C33 cells. This corresponded to a time when host DNA synthesis was reduced by 50%, and lagged by >/=1 h, the onset of viral DNA synthesis. To determine the basis for increased nuclear E2F complexes, we investigated the effects of virus infection on the intracellular distribution of the E2F-dependent DNA binding complexes and their protein constituents. Western blot analyses of whole cell extracts revealed that amounts of E2F4, E2F1, DP1, and p107 remained unchanged after infection of C33 cells. Analysis of cytoplasmic and nuclear fractions, however, revealed that cytoplasmic E2F4 decreased and nuclear E2F4 increased. This correlated with a loss of cytoplasmic E2F DNA-binding activity and a corresponding increase in nuclear DNA-binding activity. Concomitant with its redistribution, the apparent molecular weight of total and p107-associated E2F4 increased, at least partially as a result of protein phosphorylation. Increased nuclear E2F-pRB in U2-OS cells was accompanied by the conversion of pRB from a hyper- to a hypophosphorylated state. Infection of U2-OS cells with viral mutants indicated that viral protein IE ICP4 was necessary for the decrease in cytoplasmic E2F-p107, and that viral protein DE ICP8 was required for nuclear accumulation of p107-E2F. In contrast, ICP8 was not required for accumulation of E2F-pRB. These results indicate that the increase in E2F-p107 may be explained by the redistribution and modification of E2F4 and the increase in E2F-pRB by modification of pRB.     

Expression of cell-cycle-regulating transcription factor E2F-1 in colorectal carcinomas.

The expression of E2F-1 in human colorectal carcinomas was examined immunohistochemically, and the correlation of E2F-1 expression with clinicopathological findings and with the expression of p27(Kip1) was analyzed to elucidate the role of E2F-1 in the development and progression of colorectal carcinomas. In nonneoplastic mucosa, a small number of epithelial cells in the proliferative zone were weakly positive for E2F-1. Weak expression of E2F-1 was detected in many adenoma cells. Most of the colorectal carcinomas expressed E2F-1 at various levels, and strong expression of E2F-1 was detected in 56% (49/88) of the cases. There was no correlation between the expression of E2F-1 and any clinicopathological parameters such as tumor stage, depth of tumor invasion and lymph node metastasis. Reduced expression of p27(Kip1) was confirmed to be significantly correlated with deep tumor invasion and presence of metastasis. No correlation was evident between overexpression of E2F-1 and reduced p27(Kip1) expression.     

Mapping of the domains required for decay acceleration activity of the human factor H-like protein 1 and factor H

The human factor H-like protein 1 (FHL-1) is composed of seven repetitive elements (short consensus repeats; SCR) that are identical in sequence to the seven N-terminal SCR of complement factor H. We show that the FHL-1 protein has decay acceleration activity in that it can dissociate C3/C5-convertases bound to the surface of sheep red blood cells. The same activity was also determined for factor H. However, compared to FHL-1, factor H was more efficient in decay acceleration, as about 100-fold less protein was required for a 50% inhibition of activity. The domain required for decay accelerating activity of FHL-1 and factor H was mapped by the use of recombinant fragments. FHL-1 and a series of truncated forms of the protein were expressed in the baculovirus system. Recombinant FHL-1 and all mutants which include SCR 1–4 were functionally active. These four N-terminal SCR are essential and sufficient for activity, as deletion mutants which lack SCR 1 or SCR 4 showed no activity. These results demonstrate that FHL-1 and factor H have identical and overlapping regulatory functions in the complement system and that the domain required for this activity is located in the overlapping region of both proteins within the N-terminal four SCR.     

E1A genes of adenovirus type 2 and type 5 are expressed at different levels

Summary. Adenoviruses are an extensively studied system for modeling oncogenesis and for experimental cancer therapy. The most commonly analyzed virus types are 2 and 5, and little distinction has been made between them in past studies. Adenoviruses used for therapeutic purposes are frequently hybrids between these types, including the prototype dl1520/Onyx015. We tested the replication of the wild-type viruses WtD (a hybrid of the type 2 E1 region and type 5) and dl309 (type 5) in comparison with the mutants dl1520 (hybrid) and dl338 (type 5), the latter two lacking part of the E1B-55 kDa coding region. We found that the hybrid viruses replicated with considerably lower efficiency than their type 5 counterparts in H1299 cells (dl309:WtD = 3–4, dl338:dl1520 > 10). Moreover, adenovirus type 2 E1A expression from the hybrid viruses was strongly reduced in comparison to adenovirus type 5 E1A, as revealed by immunoblot analysis and RT-PCR, providing a potential explanation for the differences in virus yield. Differential E1A expression levels need to be taken into account for the construction of effective therapeutic viruses and when studying viral transformation.