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.     

E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.     

Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells.

In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed cells.     

Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions.

We have identified a cellular enhancer-binding protein, present in nuclear extracts prepared from human and rodent cells, that binds to the adenovirus E1A enhancer element I sequence. The factor has been termed EF-1A, for enhancer-binding factor to the E1A core motif. EF-1A was found to bind to two adjacent, related sequence motifs in the E1A enhancer region (termed sites A and B). The binding of EF-1A to these adjacent sites, or to synthetic dimerized sites of either motif, was cooperative. The cooperative binding of EF-1A to these sites was not subject to strict spacing constraints. EF-1A also bound to related sequences upstream of the E1A enhancer region and in the polyomavirus and adenovirus E4 enhancer regions. The EF-1A-binding region in the E1A enhancer stimulated expression of a linked gene in human 293 cells when multimerized. Based on the contact sites for EF-1A binding determined by chemical interference assays, this protein appears to be distinct from any previously characterized nuclear binding protein.     

Adenovirus early region 1A modulation of interferon antiviral activity.

Human adenovirus type 5 (Ad5) is a DNA virus which replicates as efficiently in human A549 cells treated with human interferon-alpha 2 (IFN) as in untreated cells. Vesicular stomatitis virus (VSV), on the other hand, is a negative-strand RNA virus which is very sensitive to the effects of IFN treatment in A549 cells. The IFN-mediated inhibition of VSV replication was not observed in cells coinfected with Ad5. Abrogation of IFN-mediated antiviral activity was maximal when Ad5 infection preceded VSV infection by at least 36 h, but did not require adenovirus DNA synthesis for manifestation. Coinfection experiments with VSV and deletion variants of adenovirus demonstrated that neither virus-associated RNA synthesis nor expression of adenovirus early regions E1B, E2A, E3, or E4 are required for abrogation of IFN-mediated inhibition of VSV replication. However, expression of early region E1A was essential, suggesting that E1A products can modulate, either directly or indirectly, IFN activity in adenovirus-infected cells.     

Human adenovirus-host cell interactions: comparative study with members of subgroups B and C.

Host cell interactions of human adenovirus serotypes belonging to subgroups B (adenovirus type 3 [Ad3] and Ad7) and C (Ad2 and Ad5) were comparatively analyzed at three levels: (i) binding of virus particles with host cell receptors; (ii) cointernalization of macromolecules with adenovirions; and (iii) adenovirus-induced cytoskeletal alterations. The association constants with human cell receptors were found to be similar for Ad2 and Ad3 (8 x 10(9) to 9 x 10(9) M-1), and the number of receptor sites per cell ranged from 5,000 (Ad2) to 7,000 (Ad3). Affinity blottings, competition experiments, and immunofluorescence stainings suggested that the receptor sites for adenovirus were distinct for members of subgroups B and C. Adenovirions increased the permeability of cells to macromolecules. We showed that this global effect could be divided into two distinct events: (i) cointernalization of macromolecules and virions into endocytotic vesicles, a phenomenon that occurred in a serotype-independent way, and (ii) release of macromolecules into the cytoplasm upon adenovirus-induced lysis of endosomal membranes. The latter process was found to be type specific and to require unaltered and infectious virus particles of serotype 2 or 5. Perinuclear condensation of the vimentin filament network was observed at early stages of infection with Ad2 or Ad5 but not with Ad3, Ad7, and noninfectious particles of Ad2 or Ad5, obtained by heat inactivation of wild-type virions or with the H2 ts1 mutant. This phenomenon appeared to be a cytological marker for cytoplasmic transit of infectious virions within adenovirus-infected cells. It could be experimentally dissociated from vimentin proteolysis, which was found to be serotype dependent, occurring only with members of subgroup C, regardless of the infectivity of the input virus.