E1B Gene Products Research Articles

Low-Level Expression of the E1B 20-Kilodalton Protein by Adenovirus 14p1 Enhances Viral Immunopathogenesis.

Adenovirus 14p1 (Ad14p1) is an emergent variant of Ad serotype 14 (Ad14) that has caused increased severity of respiratory illnesses during globally distributed outbreaks, including cases of acute respiratory distress syndrome and death. We found that human cell infection with Ad14p1 results in markedly decreased expression of the E1B 20-kilodalton (20K) protein compared to that with infection with wild-type (wt) Ad14. This reduced Ad14p1 E1B 20K expression caused a loss-of-function phenotype of Ad-infected cell corpses that, in contrast to cells infected with wt Ad14, either failed to repress or increased NF-κB-dependent, proinflammatory cytokine responses of responder human alveolar macrophages. A small-animal model of Ad14-induced lung infection was used to test the translational relevance of these in vitro observations. Intratracheal infection of Syrian hamsters with Ad14p1 caused a marked, patchy bronchopneumonia, whereas hamster infection with wt Ad14 caused minimal peribronchial inflammation. These results suggest that this difference in E1B 20K gene expression during Ad14p1 infection and its modulating effect on the interactions between Ad14-infected cells and the host innate immune response could explain the increased immunopathogenic potential and associated increase in clinical illness in some people infected with the Ad14p1 outbreak strain.IMPORTANCE We previously reported that Ad-infected human cells exhibit E1B 19K-dependent repression of virally induced, NF-κB-dependent macrophage cytokine responses (J. R. Radke, F. Grigera, D. S. Ucker, and J. L. Cook, J Virol 88:2658-2669, 2014, http://dx.doi.org/10.1128/JVI.02372-13). The more virulent, emergent strain of Ad14, Ad14p1, causes increased cytopathology in vitro, which suggested a possible E1B 20K defect. Whether there is a linkage between these observations was unknown. We show that there is markedly reduced expression of E1B 20K in Ad14p1-infected human cells and that this causes an increased proinflammatory cytokine response of human alveolar macrophages and more severe inflammatory lung disease in infected hamsters. This is the first evidence of a clinical relevance of differential expression of the small Ad E1B gene product. The results suggest that there is a low, critical threshold of E1B 19/20K expression that is needed for viral replication and infection transmission but that a higher level of E1B 19/20K expression is required for the usual repression and control of the Ad-triggered host innate immune response.

775. An Novel Adenoviral E1 Complementary Cell for Eliminating Replication Competent Adenovirus (RCA) during Viral Production

Production of recombinant adenoviruses from 293 cells and other recently developed adenoviral E1-complementary cell lines have the potential to produce low levels of replication competent adenoviruses (RCA) derived from cellular DNA recombination. In most E1 complementary cells, varying lengths of the adenoviral E1A and E1B coding regions and their transcriptional regulatory structures have been used to generate the cell lines. A 3Kb fragment of the viral genome containing both the E1A and E1B coding regions (nts 560 to 3510 of adenovirus 5) is essential in developing such cell lines. This leaves open the possibility for cellular-derived E1 DNA to undergo homologous recombination with both E1-deleted replication defective and E1-modified replication competent adenoviral vectors. In order to mitigate the potential for cell-derived homologous recombination during adenovirus production, we applied a different strategy to generate E1-complementary cells. The coding sequences for adenovirus E1A and E1B were cloned into separate retroviral vectors derived from Moloney Murine Leukemia Virus (MMLV). E1A or E1B expressing transient retroviral supernatants were separately prepared from these vectors and used to co-infect cells. The A549 human lung carcinoma cell line was co-infected by the supernatants containing the MMLV-E1A and MMLV-E1B vectors. Limiting dilution and functional screens led to the isolation of adenoviral E1-complementary cell lines. These cells support the replication of E1-defective adenoviral vectors and express the E1A and E1B gene products as demonstrated by Western blot. Of note, instead of a continuous strand of adenoviral DNA, the E1A and E1B genes are separately integrated as two smaller DNA fragments. This arrangement is designed to minimize the chance of RCA generation derived from the recombination of viral DNA with cellular DNA. In addition to supporting the replication of E1-defective adenoviral vectors, further characterization of these cell lines demonstrated that they complement the replication of E1-modified replicating adenoviral vectors in which the E1 genes are under the control of tissue-specific, transcriptional regulatory elements. Comparisons of 293, PerC6, and the A549-derived E1-complementary cell lines suggest equivalent viral production. This complementation ability was stable over 10 weeks in culture and multiple passages of the cell lines. Preliminary RCA assessment suggests that these cells are superior to 293 cells and other cells in reducing the production of RCA.

Adenovirus and cell cycle control.

Adenovirus infection of quiescent cells induces transition from G0 or G1 into the S phase of the cell cycle and allows cellular proliferation. This is beneficial for the virus since cells in S phase provide optimal conditions for viral replication. Adenovirus E1A, E1B and E4 gene products contribute to cell cycle deregulation. E1A proteins inactivate the pRb checkpoint, allowing the E2F transcription factor to activate genes involved in nucleotide metabolism and DNA replication, which are required in S phase. E1A also interacts with transcriptional modulators, including histone acetyltransferases, histone deacetylases, and other chromatin remodeling factors. These interactions affect transcription of several cellular and viral genes, some of which are involved in cell cycle regulation. Cell cycle deregulation by E1A results in stabilization and accumulation of p53. To prevent cell cycle arrest and apoptosis that would be triggered by p53, the adenovirus E1B and E4orf6 gene products employ various mechanisms to inactivate the tumor suppressor. Additional E4 gene products also interact with and modulate cell cycle regulators. Cell cycle checkpoints targeted by adenovirus proteins are often compromised in human tumors as well. Thus, understanding the interactions between adenovirus and the cell cycle has facilitated the generation of adenovirus mutants, which can replicate only in cells with inactivated checkpoints. Such "oncolytic" viruses are being tested for their ability to specifically replicate in and lyse cancer cells.

"Hit-and-run" transformation by adenovirus oncogenes.

According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the "hit-and-run" hypothesis claims that viruses can mediate cellular transformation through an initial "hit," while maintenance of the transformed state is compatible with the loss ("run") of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy.

Adenovirus E1A oncogene expression in tumor cells enhances killing by TNF-related apoptosis-inducing ligand (TRAIL).

Expression of the adenovirus serotype 5 (Ad5) E1A oncogene sensitizes cells to apoptosis by TNF-alpha and Fas-ligand. Because TNF-related apoptosis-inducing ligand (TRAIL) kills cells in a similar manner as TNF-alpha and Fas ligand, we asked whether E1A expression might sensitize cells to lysis by TRAIL. To test this hypothesis, we examined TRAIL-induced killing of human melanoma (A2058) or fibrosarcoma (H4) cells that expressed E1A following either infection with Ad5 or stable transfection with Ad5-E1A. E1A-transfected A2058 (A2058-E1A) or H4 (H4-E1A) cells were highly sensitive to TRAIL-induced killing, but Ad5-infected cells expressing equally high levels of E1A protein remained resistant to TRAIL. Infection of A2058-E1A cells with Ad5 reduced their sensitivity to TRAIL-dependent killing. Therefore, viral gene products expressed following infection with Ad5 inhibited the sensitivity to TRAIL-induced killing conferred by transfection with E1A. E1B and E3 gene products have been shown to inhibit TNF-alpha- and Fas-dependent killing. The effect of these gene products on TRAIL-dependent killing was examined by using Ad5-mutants that did not express either the E3 (H5dl327) or E1B-19K (H5dl250) coding regions. A2058 cells infected with H5dl327 were susceptible to TRAIL-dependent killing. Furthermore, TRAIL-dependent killing of A2058-E1A cells was not inhibited by infection with H5dl327. Infection with H5dl250 sensitized A2058 cells to TRAIL-induced killing, but considerably less than H5dl327-infection. In summary, expression of Ad5-E1A gene products sensitizes cells to TRAIL-dependent killing, whereas E3 gene products, and to a lesser extent E1B-19K, inhibit this effect.

Esophagogastric adenocarcinoma in an E1A/E1B transgenic model involves p53 disruption

We studied tumorigenesis and p53 immunostaining in a murine transgenic model introducing E1A/E1B under the control of the mouse mammary tumor virus—long terminal repeat (MMTV-LTR) promoter in which adenocarcinoma occurs at the squamocolumnar junction in the foregut, predominantly in males, and at no other site. Mutations of p53 are frequent in human esophageal adenocarcinoma and the E1B gene product interferes with p53-mediated apoptosis, inhibiting tumor suppression at the G 1/S checkpoint. Transgenic animals were generated utilizing a purified linear 6.7 kb fragment of plasmid DNA containing MMTV-LTR/E1A/E1B and were confirmed by dot blot hybridization of tail DNA to 32P-labeled E1A/E1B probe and polymerase chain reaction (PCR) amplification of E1A. Transgenic and control animals were observed for morbidity and weight changes. Eleven of 45 animals were transgenic (24% efficiency) with an estimated 5 to 57 copies of the gene per genome. Profound weight loss (20%) led to sacrifice or death of one of five females (at 12 weeks) and four of six males (at 16 to 17 weeks). Grossly visible tumors (2 to 10 mm) were note in the forestomach at the visible margin between the proximal (squamous-lined) stomach and the distal glandular stomach. Histologic sections confirmed adenocarcinoma arising in each case at the squamocolumnar junction with glandular formation, pleomorphism, and frequent mitotic figures. Immunostaining was positive for p53 indicating accumulation of mutated or altered p53 protein. E1A/E1B transgenic animals developed macroscopic and microscopic adenocarcinoma at the squamocolumnar junction, which corresponds to adenocarcinoma at the human esophagogastric junction. Disruption of p53 was present in the transgenic model as in the human cancer.

Transforming potential of the adenovirus type 5 E4orf3 protein.

Previous observations that the adenovirus type 5 (Ad5) E4orf6 and E4orf3 gene products have redundant effects in viral lytic infection together with the recent findings that E4orf6 possesses transforming potential prompted us to investigate the effect of E4orf3 expression on the transformation of primary rat cells in combination with adenovirus E1 oncogene products. Our results demonstrate for the first time that E4orf3 can cooperate with adenovirus E1A and E1A plus E1B proteins to transform primary baby rat kidney cells, acting synergistically with E4orf6 in the presence of E1B gene products. Transformed rat cells expressing E4orf3 exhibit morphological alterations, higher growth rates and saturation densities, and increased tumorigenicity compared with transformants expressing E1 proteins only. Consistent with previous results for adenovirus-infected cells, the E4orf3 protein is immunologically restricted to discrete nuclear structures known as PML oncogenic domains (PODs) in transformed rat cells. As opposed to E4orf6, the ability of E4orf3 to promote oncogenic cell growth is probably not linked to a modulation of p53 functions and stability. Instead, our results indicate that the transforming activities of E4orf3 are due to combinatorial effects that involve the binding to the adenovirus 55-kDa E1B protein and the colocalization with PODs independent from interactions with the PML gene product. These data fit well with a model in which the reorganization of PODs may trigger a cascade of processes that cause uncontrolled cell proliferation and neoplastic growth. In sum, our results provide strong evidence for the idea that interactions with PODs by viral proteins are linked to oncogenic transformation.

Adenovirus E1B 19,000-molecular-weight protein activates c-Jun N-terminal kinase and c-Jun-mediated transcription.

Adenovirus E1B proteins (19,000-molecular-weight [19K] and 55K proteins) inhibit apoptosis and cooperate with adenovirus E1A to induce full oncogenic transformation of primary cells. The E1B 19K protein has previously been shown to be capable of activating transcription; however, the underlying mechanisms are unclear. Here, we show that adenovirus infection activates the c-Jun N-terminal kinase (JNK) and that the E1B gene products are necessary for adenovirus to activate JNK. In transfection assays, we show that the E1B 19K protein is sufficient to activate JNK and can strongly induce c-Jun-dependent transcription. Mapping studies show that the C-terminal portion of E1B 19K is necessary for induction of c-Jun-mediated transcription. Using dominant-negative mutants of several kinases upstream of JNK, we show that MEKK1 and MKK4, but not Ras, are involved in the induction of JNK activity by adenovirus infection. The same dominant-negative kinase mutants also block the ability of E1B 19K to induce c-Jun-mediated transcription. Taken together, these results suggest that E1B 19K may utilize the MEKK1-MKK4-JNK signaling pathway to activate c-Jun-dependent transcription and demonstrate a novel, kinase-activating activity of E1B 19K that may underlie its ability to regulate transcription.

Regulation of viral and cellular gene expression by E1A proteins encoded by the oncogenic adenovirus type 12.

For more than 15 years human adenoviruses (Ad) have been a powerful tool for studying cellular processes such as regulation of gene expression, alternative splicing, polyadenylation, and replication. Especially the analysis of viral proteins encoded by early region 1A (E1A), which was shown to regulate transcription, has given many insights into how DNA viruses regulate their own and cellular gene expression. Moreover, E1A proteins have been the subject of extensive studies because of their ability to act as oncoproteins that cooperate with the adenovirus E1B gene products to transform rodent cells in culture and, in case of the oncogenic adenoviruses (e.g., Ad12), to induce tumors in animals (Gallimore et al. 1974; Graham et al. 1974a, b; Houweling 1980; Jochemsen et al. 1982). The ability to promote oncogenic transformation and transcriptional regulation appear to be distinct activities of the E1A polypeptides (for review see Moran and Mathews 1987). In oncogenic transformation, protein functions of region E1A are necessary to immortalize primary cells, whereas functions of region E1B are essential to obtain a fully transformed phenotype. The functions of region E1B can be substituted by specific cellular gene products, e.g., activated Ha-ras (Byrd et al. 1988; Ruley 1983). The reasons for the difference in oncogenicity of variant adenovirus serotypes are not yet understood.KeywordsAdenovirus TypeCellular Gene ExpressionCellular Transcription FactorHuman Adenovirus TypeRetinoblastoma Gene ProductThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome.

Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome.

Genetic analysis of carcinogen enhancement of type 5 adenovirus transformation of cloned Fischer rat embryo fibroblast cells.

Pretreatment of CREF cells with methyl methanesulfonate (MMS) before infection with the host-range cold-sensitive type 5 adenovirus (Ad5) mutant H5hr1 results in a dose-dependent carcinogen enhancement of viral transformation (CET). The properties of CET observed with H5hr1, which include both an MMS dose-dependent enhancement in the number of transformed foci and an increase in transformation frequency after correction for cell toxicity, are not observed in carcinogen-pretreated wild-type (wt) Ad5 (H5wt)-infected CREF cells. This study was conducted to determine the role of the viral E1A and E1B transforming genes of H5hr1 in mediating the unique CET phenotype of H5hr1. Coinfection of MMS-pretreated CREF cells with H5wt or H5sub309 (which displays a wt Ad5 phenotype) and H5hr1 resulted in a suppression of the unique CET phenotype that was directly related to the multiplicity of infection with wt Ad5. Suppression of the unique H5hr1 CET phenotype was also apparent in MMS-pretreated CREF cells coinfected with H5hr1 and an Ad5 mutant expressing either a wt 13S E1A-encoded 289 amino-acid (aa) protein and an intact wt E1B gene or a wt 13S E1A-encoded 289-aa protein and a 22S E1B-encoded 495-aa protein. In contrast, the unique H5hr1 CET phenotype was not suppressed in MMS-pretreated CREF cells coinfected with H5hr1 and Ad5 or Ad2 mutants expressing either a wt 12S E1A-encoded 243-aa protein and both wt E1B gene products or an intact wt E1A gene and a wt E1B 13S-encoded 175-aa protein. That genetic changes in both the E1A and E1B viral regions of H5hr1 were required to induce the unique CET phenotype was also indicated by the inability of a recombinant Ad5 containing the 0-4.5 map-unit region of H5hr1 and the 4.5-100 map-unit region of H5sub309 to display the H5hr1 unique CET phenotype. Direct confirmation of the requirement for both gene regions of H5hr1 to mediate its unique CET was obtained by generating CREF cells stably expressing a wt Ad5 E1A 13S-encoded 289-aa protein and a wt E1B 22S-encoded 495-aa protein. In these CREF transformants (which displayed a CREF-like morphology), transformation by H5hr1 was not reduced, but the unique CET phenotype after MMS pretreatment was eliminated. These results suggest that alterations in both the 13S-encoded E1A and 22S-encoded E1B gene products of H5hr1 contribute to its unique CET.

Activation of the mouse proliferating cell nuclear antigen gene promoter by adenovirus type 12 E1A proteins.

A plasmid carrying the 5′‐flanking region (– 1584 to + 47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5‐ to 9‐fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of El A resides in a common region(s) of 13S and 12S El A products. The major target region of El A was mapped within the 68 base‐pair region (‐21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2P, although the upstream region (–83 to – 21) including ATF(CREB)‐binding consensus had an additional effect in the transactivation.

Activation of the mouse DNA polymerase β gene promoter by adenovirus type 12 E1A proteins

A plasmid carrying the 5'-flanking region (-1852 to +33 with respect to the transcription initiation site) of the mouse DNA polymerase beta gene fused with the chloramphenicol acetyltransferase (CAT) gene was cotransfected into mouse N18TG2 cells with adenovirus type 12 E1 genes-expressing plasmids. Expression of E1A gene products resulted in the elevation of the CAT expression by 3 to 7 folds, but that of E1B gene product was much less effective. RNase protection analysis revealed that the activation by E1A was at the transcription process. Both the 13S E1A and the 12S E1A activated the DNA polymerase beta gene promoter, indicating that the activation domain of E1A is in a common region(s) of 13S and 12S E1A products. The major target sequence of E1A was mapped within the 10 base pair-region (-30 to -20) of the DNA polymerase beta gene promoter, which overlapped with the palindromic sequence known as the ATF(CREB)/E4F-binding consensus. The results suggest that the palindromic sequence is essential for E1A-induced transcriptional activation of the mouse DNA polymerase beta gene.

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.

Fastidious human adenovirus type 40 can propagate efficiently and produce plaques on a human cell line, A549, derived from lung carcinoma

Human adenovirus type 40 (Ad40) cannot propagate in conventional established human cell lines such as KB or HeLa cells. However, it has been shown that Ad40 DNA replicates in KB18 cells which express Ad2 E1B genes, suggesting that Ad40 is defective in the E1B gene function in KB or HeLa cells. We show here that Ad40 can propagate and produce plaques on A549 cells which do not contain Ad E1B genes. Our experiments show that the levels of replication of Ad40 DNA and production of infectious Ad40 virus in A549 cells are the same as or higher than those in 293 or KB18 cells. Dot blot analysis shows that the levels of Ad40 E1A and E1B mRNAs expressed in A549 cells at early to intermediate times postinfection are at least 10-fold higher than those in KB or KB18 cells. Northern (RNA) blot analysis shows that large E1B mRNA species (approximately 24S to 26S) are synthesized prior to the onset of DNA replication in A549 cells. No E1B mRNA species are synthesized in KB or KB18 cells at early times postinfection, and no differences in the expression of E1B mRNAs are seen between KB and KB18 cells. The experiment suggests that A549 cells have a cellular factor(s) which activates Ad40 E1B mRNA synthesis and that the E1B mRNA synthesis helps Ad40 propagation. In contrast, Ad40 can propagate in KB18 cells by using Ad2 E1B gene products that are constitutively expressed in this cell line. Furthermore, this result shows that Ad40 cannot propagate in KB cells because of the failure in the expression of E1B genes at early times postinfection.

Suppression of the progression phenotype in somatic cell hybrids occurs in the absence of altered adenovirus type 5 gene expression.

In the present study we have analyzed the genetic regulation of increased expression of transformation-associated traits, a process termed progression, in adenovirus type 5 (Ad5)-transformed secondary rat embryo cells. Somatic cell hybrids were formed between a highly progressed neomycin-resistant Ad5-transformed cloned cell line (E11-NMTneo) and an untransformed chloramphenicol-resistant rat embryo fibroblast cell line (CREFcap). Parental E11-NMTneo cells grew with high efficiency in agar, exhibited reduced 125I-epidermal growth factor (EGF) binding, and were tumorigenic in nude mice. Parental CREFcap cells exhibited phenotypes opposite to those of E11-NMTneo cells. A high proportion (84%) of the presumptive hybrid cell types obtained after fusion and genetic selection (G418 and chloramphenicol) displayed a flat morphological phenotype intermediate between CREFcap and E11-NMTneo cells, suggesting that a trans-dominant extinction phenomenon had occurred. Two hybrids with a round morphology (R), which still exhibited the progressed phenotype, and two hybrids with a flat morphology (F), which had lost the progressed phenotype, were chosen for detailed analysis. Both R hybrids grew efficiently in agar, exhibited low 125I-EGF binding, and were tumorigenic in nude mice, whereas both F hybrids grew poorly in agar, displayed increased 125I-EGF binding in comparison with E11-NMTneo and R hybrids, and were nontumorigenic in nude mice. An analysis of the viral DNA integration patterns and the rates of transcription, steady-state mRNA accumulation, and relative levels of the Ad5 E1A and E1B gene products revealed no differences among the parental and hybrid cells. These studies indicate that normal CREF cells may contain a suppressor gene(s) which can inhibit the expression of specific traits of the progression phenotype in Ad5-transformed cells and that this suppression is not associated with changes in the expression of Ad5 transforming genes.

Suppression of the Progression Phenotype in Somatic Cell Hybrids Occurs in the Absence of Altered Adenovirus Type 5 Gene Expression

In the present study we have analyzed the genetic regulation of increased expression of transformation-associated traits, a process termed progression, in adenovirus type 5 (Ad5)-transformed secondary rat embryo cells. Somatic cell hybrids were formed between a highly progressed neomycin-resistant Ad5-transformed cloned cell line (E11-NMTneo) and an untransformed chloramphenicol-resistant rat embryo fibroblast cell line (CREFcap). Parental E11-NMTneo cells grew with high efficiency in agar, exhibited reduced 125I-epidermal growth factor (EGF) binding, and were tumorigenic in nude mice. Parental CREFcap cells exhibited phenotypes opposite to those of E11-NMTneo cells. A high proportion (84%) of the presumptive hybrid cell types obtained after fusion and genetic selection (G418 and chloramphenicol) displayed a flat morphological phenotype intermediate between CREFcap and E11-NMTneo cells, suggesting that a trans-dominant extinction phenomenon had occurred. Two hybrids with a round morphology (R), which still exhibited the progressed phenotype, and two hybrids with a flat morphology (F), which had lost the progressed phenotype, were chosen for detailed analysis. Both R hybrids grew efficiently in agar, exhibited low 125I-EGF binding, and were tumorigenic in nude mice, whereas both F hybrids grew poorly in agar, displayed increased 125I-EGF binding in comparison with E11-NMTneo and R hybrids, and were nontumorigenic in nude mice. An analysis of the viral DNA integration patterns and the rates of transcription, steady-state mRNA accumulation, and relative levels of the Ad5 E1A and E1B gene products revealed no differences among the parental and hybrid cells. These studies indicate that normal CREF cells may contain a suppressor gene(s) which can inhibit the expression of specific traits of the progression phenotype in Ad5-transformed cells and that this suppression is not associated with changes in the expression of Ad5 transforming genes.

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.