E1B Oncogenes Research Articles

Viral oncogenes, noncoding RNAs, and RNA splicing in human tumor viruses.

Viral oncogenes are responsible for oncogenesis resulting from persistent virus infection. Although different human tumor viruses express different viral oncogenes and induce different tumors, their oncoproteins often target similar sets of cellular tumor suppressors or signal pathways to immortalize and/or transform infected cells. Expression of the viral E6 and E7 oncogenes in papillomavirus, E1A and E1B oncogenes in adenovirus, large T and small t antigen in polyomavirus, and Tax oncogene in HTLV-1 are regulated by alternative RNA splicing. However, this regulation is only partially understood. DNA tumor viruses also encode noncoding RNAs, including viral microRNAs, that disturb normal cell functions. Among the determined viral microRNA precursors, EBV encodes 25 from two major clusters (BART and BHRF1), KSHV encodes 12 from a latent region, human polyomavirus MCV produce only one microRNA from the late region antisense to early transcripts, but HPVs appears to produce no viral microRNAs.

Regulation of Apoptosis by Adenovirus E1A and E1B Oncogenes

Adenovirus E1A promotes apoptosis by interacting with and inhibiting negative regulators of cell cycle control. Binding of E1A to, and inhibition of, the transcriptional coadaptor p300 promotes accumulation of the p53 tumor suppressor protein which induces apoptosis. By inhibiting p300, E1A prevents the transcriptional activation ofmdm-2,the product of which interacts with and promotes the degradation of p53. Thus the E1A–p300 interaction disables the negative feedback loop to control p53 levels, which left unrestrained, cause apoptosis rather than growth arrest. The E1B 19K protein functions analogously to Bcl-2 to inhibit apoptosis by E1A, p53, and multiple other stimuli. The E1B 19K protein functions by at least two independent mechanisms to inhibit apoptosis. First, the E1B 19K protein binds to the pro-apoptotic Bax protein to prevent loss of mitochondrial membrane potential, caspase activation, and apoptosis. Second, the E1B 19K protein inhibits caspase interaction by interfering with the function of adaptor molecules such as FADD and Ced-4 that interact with and activate caspases. By inhibiting FADD-dependent activation of the caspase FLICE, the E1B 19K protein can disable both the TNF-α- and the Fas-mediated death signaling pathways which play an important role in immune surveillance against virus infection and cancer. The E1B 19K protein binds to Ced-4, and presumably mammalian Ced-4 homologues, and thereby prevents caspase activation. Thus, the study of the mechanism of regulation of apoptosis by the adenovirus transforming proteins has revealed important regulatory steps in death signaling pathways.

Mapping of adenovirus 5 E1A domains responsible for suppression of neu-mediated transformation via transcriptional repression of neu.

Overexpression of neu (also known as c-erbB-2 or HER-2) commonly occurs in human cancer and is also known to enchance tumor metastasis and chemoresistance. Our earlier reports showed that the adenovirus 5 E1A can suppresss the neu-mediated transformation by repression of neu. Thus, E1A has the potential to be used as a therapeutic agent against the neu-overexpressing human cancers. However, a serious concern to this approach is that E1A is also capable of immortalizing primary culture cells and can co-operate with ras or E1B oncogenes to transform them. The E1A CR2 domain (amino acid residues 120 to 140) necessary for binding to RB is believed to be required for this oncogenic function. Here, we report that deletion of CR2 region did not affect E1A's capability to repress neu. Interestingly, deletion of the amino acid residues 4 to 25 or 40 to 80 completely disrupted E1A-mediated neu repression. By deleting the amino acid residues from 81 to 185, we have successfully generated a mini-E1A mutant that was sufficient to inhibit neu promoter activity and suppress neu-mediated transformation. The mini-E1A mutant does not contain the CR2 domain that is crucial for RB binding and immortalization, and hence, may serve as a more selective tumor suppressor, and a safer therapeutic agent. It may also be a useful tool to further investigate the molecular mechanism(s) of neu overexpression and E1A-mediated transcriptional repression in cancer cells.

Oncogenic potential of the adenovirus E4orf6 protein.

The group C adenovirus E4orf6 protein has previously been shown to bind to the p53 cellular tumor suppressor protein and block its ability to activate transcription. Here we show that the E4orf6 protein blocks the induction of p53-mediated apoptosis when AT6 cells, which harbor a temperature-sensitive p53, are shifted to the permissive temperature. The E4orf6 protein does not, however, prevent the induction of apoptosis in p53-deficient H1299 cells by treatment with tumor necrosis factor alpha and cycloheximide. The E4orf6 protein also cooperates with the adenovirus E1A protein to transform primary baby rat kidney cells, and it cooperates with the adenovirus E1A plus E1B 19-kDa and E1B 55-kDa proteins to increase the number of baby rat kidney cell transformants and enhance the rate at which they arise. The level of p53 is substantially reduced in transformed cells expressing the E4orf6 protein in comparison to adenovirus transformants lacking it. The E4orf6 gene also accelerates tumor formation when transformed baby rat kidney cells are injected subcutaneously into the nude mouse, and it converts human 293 cells from nontumorigenic to tumorigenic in nude mice. In addition to the well-studied E1A and E1B oncogenes, group C adenoviruses harbor a third oncogene, E4orf6, which functions in some respects similarly to the E1B oncogene.

Function of the adenovirus E1B oncogene in infected and transformed cells

Adenovirus infection and E1A gene expression stimulates cellular proliferation as a mechanism to facilitate virus replication. Programmed cell death (apoptosis) is the cellular response to this deregulation of growth control by E1A during viral infection and neoplastic transformation. To combat the suicidal elimination of virus infected cells by apoptosis, adenovirus has evolved a mechanism to disengage the apoptotic program of the cell. This anti-apoptotic function is encoded within the adenovirus E1B 19 kDa and 55 kDa gene products. Both viral products encoded by E1B act at independent and overlapping points in the cell death process to ensure that the premature death of the host cell does not take place and that viral infection can progress to completion. The E1B 55K protein functions as an anti-apoptotic gene product by direct physical interference with the p53 tumor suppressor protein, whereas the E1B 19K protein acts to inhibit p53-dependent and probably p53-independent apoptosis by a mechanism that resembles that of the human bcl-2 protooncogene.

Regulation of Apoptosis by the Transforming Genes of the DNA Tumor Virus Adenovirus

The human DNA tumor virus adenovirus encodes two transforming genes, the E1A and E1B oncogenes, which cooperate to oncogenically transform primary rodent cells. The E1A products efficiently stimulate cell proliferation, but fail to transform cells due to the induction of programmed cell death (apoptosis). Expression of either the E1B oncogene, or the human bcl-2 proto-oncogene, blocks E1A-associated apoptosis to produce transformation with high frequency. Thus, induction of proliferation by E1A must be coupled to suppression of an intrinsic cell suicide pathway for the efficient transformation of primary cells.The E1B oncogene encodes the unique 19-kDa and 55-kDa proteins, both of which independently suppress apoptosis and greatly enhance transformation by E1A. Suppression of apoptosis by the E1B 19-kDa protein is required not only in transformation of rodent cells with E1A, but also in adenovirus-infected human cells, where it sustains cell viability to maximize virus production. The E1B 19-kDa protein h...

Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B.

Transformation of primary rodent cells by the adenovirus E1A and E1B oncogenes is a two-step process, where E1A-dependent induction of proliferation is coupled to E1B-dependent suppression of programmed cell death (apoptosis). The E1B gene encodes two distinct transforming proteins, the 19K and 55K proteins, both of which independently cooperate with E1A. E1B 19K or 55K protein, or the human Bcl-2 protein, functions to suppress apoptosis and thereby permits transformation with E1A. The E1B 55K protein blocks p53 tumor suppressor protein function, indicating that p53 may mediate apoptosis by E1A. In the mutant conformation, p53 blocked induction of apoptosis by E1A and efficiently cooperated with E1A to transform primary cells. When p53 was returned to the wild-type conformation, E1A+p53 transformants underwent cell death by apoptosis. This induction of apoptosis by conformational shift of p53 from the mutant to the wild-type form was inhibited by expression of the E1B 19K protein. Thus, the p53 protein may function as a tumor suppressor by initiating a cell suicide response to deregulation of growth control by E1A. E1B 19K and 55K proteins provide separate mechanisms that disable the cell suicide pathway of p53.

The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.

Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis.

The E1b oncogene of adenovirus confers cellular resistance to cytotoxicity of tumor necrosis factor and monoclonal anti-Fas antibody

The cell lines KB8, 16, and 18 are KB cells which constitutively express adenovirus type 2 (Ad2) E1a, E1a plus E1b, and E1b genes, respectively. We show here that KB18 cells are completely resistant to cytolysis by tumor necrosis factor (TNF) or anti-Fas, although KB8 and KB or KB16 are highly and moderately sensitive, respectively. The levels of receptors for TNF and anti-Fas of KB18 were almost the same as compared with those of KB or other KB-cell lines. Expression of manganous superoxide dismutase (MnSOD) mRNA in KB18 was about 20-fold higher than that in KB or KB8 cells. KB, HT29, and A673 cells infected with dl337 (an Ad5 mutant defective in E1b function) are highly sensitive to TNF or anti-Fas, although wild-type Ad2-infected cells are resistant. Our results indicate that the E1b oncogene can confer cellular resistance to cytolysis by either TNF or anti-Fas in both KB cells and adenovirus-infected human cell lines through influencing intracellular events including regulation of MnSOD genes. Furthermore, we describe how anti-Fas mimics only the cytolytic activity of TNF, whereas TNF also has many other biological activities.

Role of adenovirus E1B proteins in transformation: altered organization of intermediate filaments in transformed cells that express the 19-kilodalton protein.

Cooperation of the nuclear oncogene E1A with the E1B oncogene is required for transformation of primary cells. Expression vectors were constructed to produce the 19-kilodalton (19K) and 55K E1B proteins under the direction of heterologous promoters in order to investigate the role of individual E1B proteins in transformation. Coexpression of E1A and either the 19K or 55K E1B gene products was sufficient for the formation of transformed foci in primary rat cells at half the frequency of an intact E1B gene, suggesting that the 19K and 55K proteins function via independent pathways in transformation. Furthermore, the effects of Ha-ras and the E1B 19K gene product were additive when cotransfected with E1A, suggesting that the 19K protein functions in transformation by a mechanism independent from that of ras as well. Although expression of E1A and either E1B protein was sufficient for the subsequent growth of cells in long-term culture, the 19K protein was required to support growth in semisolid media. As the 19K protein has been shown to associate with and disrupt intermediate filaments (IFs) when transiently expressed with plasmid vectors (E. White and R. Cipriani, Proc. Natl. Acad. Sci. USA, 86:9886-9890, 1989), the organization of IFs in transformed cells was investigated. Primary rat cells transformed by plasmids encoding E1A plus the E1B 19K protein showed gross perturbations of IFs, whereas cell lines transformed by plasmids encoding E1A plus the E1B 55K protein or E1A plus Ha-ras did not. These results suggest that an intact IF cytoskeleton may inhibit anchorage-independent growth and that the E1B 19K protein can overcome this inhibition by disrupting the IF cytoskeleton.

Role of Adenovirus E1B Proteins in Transformation: Altered Organization of Intermediate Filaments in Transformed Cells That Express the 19-Kilodalton Protein

Cooperation of the nuclear oncogene E1A with the E1B oncogene is required for transformation of primary cells. Expression vectors were constructed to produce the 19-kilodalton (19K) and 55K E1B proteins under the direction of heterologous promoters in order to investigate the role of individual E1B proteins in transformation. Coexpression of E1A and either the 19K or 55K E1B gene products was sufficient for the formation of transformed foci in primary rat cells at half the frequency of an intact E1B gene, suggesting that the 19K and 55K proteins function via independent pathways in transformation. Furthermore, the effects of Ha-ras and the E1B 19K gene product were additive when cotransfected with E1A, suggesting that the 19K protein functions in transformation by a mechanism independent from that of ras as well. Although expression of E1A and either E1B protein was sufficient for the subsequent growth of cells in long-term culture, the 19K protein was required to support growth in semisolid media. As the 19K protein has been shown to associate with and disrupt intermediate filaments (IFs) when transiently expressed with plasmid vectors (E. White and R. Cipriani, Proc. Natl. Acad. Sci. USA, 86:9886-9890, 1989), the organization of IFs in transformed cells was investigated. Primary rat cells transformed by plasmids encoding E1A plus the E1B 19K protein showed gross perturbations of IFs, whereas cell lines transformed by plasmids encoding E1A plus the E1B 55K protein or E1A plus Ha-ras did not. These results suggest that an intact IF cytoskeleton may inhibit anchorage-independent growth and that the E1B 19K protein can overcome this inhibition by disrupting the IF cytoskeleton.

Specific disruption of intermediate filaments and the nuclear lamina by the 19-kDa product of the adenovirus E1B oncogene.

The 19-kDa protein encoded within the adenovirus E1B gene is essential for transformation by adenovirus and for proper regulation of viral early gene transcription. In order to investigate the biological function of the 19-kDa E1B protein, vectors were constructed to produce the 19-kDa protein in mammalian cells under the direction of heterologous promoters. Surprisingly, during transient expression, the E1B 19-kDa protein specifically associated with and disrupted the organization of intermediate filaments and the nuclear lamina, without disturbing the organization of other cytoskeletal networks. These results directly demonstrate an effect of a viral transforming protein on the cytoskeleton and suggest a role for intermediate filaments and the nuclear lamina in modulation of viral gene expression and the process of oncogenic transformation.