Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins.

Adenovirus mutants containing genetic alterations in the gene encoding the E1B 19,000-molecular-weight (19K) tumor antigen induce the degradation of host cell chromosomal DNA (deg phenotype) and enhanced cytopathic effect (cyt phenotype) after infection of HeLa and KB cells. The deg and cyt phenotypes are a consequence of viral early gene expression in the absence of the E1B 19K protein. The role of the E1A proteins in induction of the cyt and deg phenotypes was investigated by constructing E1A-E1B double mutant viruses. Viruses were constructed to express the individual E1A 13S, 12S, or 9S cDNA genes in the presence of a mutation in the gene encoding the E1B 19K tumor antigen. Expression of either the 13S or 12S E1A proteins in the absence of functional E1B 19K protein produced the deg and cyt phenotypes. In contrast, a virus which expressed exclusively the 9S E1A gene product in the absence of the E1B 19K gene product did not induce the deg and cyt phenotypes, even at high multiplicities of infection. Therefore, both the 13S and 12S E1A gene products could directly or indirectly cause the deg and cyt phenotypes during infection of HeLa cells with an E1B 19K gene mutant virus. Furthermore, the deg phenotype was found to be host cell type specific, occurring in HeLa and KB cells but not in growth-arrested human WI38 cells. These results indicate that expression of the E1A trans-activating and transforming proteins is necessary for the induction of the cyt and deg phenotypes and that host cell factors also play a role.     

Absence of an essential regulatory influence of the adenovirus E1B 19-kilodalton protein on viral growth and early gene expression in human diploid WI38, HeLa, and A549 cells.

Mutations in the gene encoding the adenovirus (Ad) early region 1B 19-kDa protein (the 19K gene) result in multiple phenotypic effects upon infection of permissive human cells. It has been reported, for example, that Ad type 2 (Ad2) and Ad5 with mutations in the 19K gene (19K-defective mutants) have a marked growth advantage compared with wild-type virus in human diploid WI38 cells (E. White, B. Faha, and B. Stillman, Mol. Cell. Biol. 6:3763-3773, 1986), and it was proposed that this host range phenotype stems from the large increase in viral early gene expression reported to occur in the mutant-infected cells. These observations gave rise to the hypothesis that the 19-kDa protein (the 19K protein) normally functions as a negative regulator of Ad early gene expression and growth. We have tested this hypothesis and find that Ad5 and Ad12 wild-type viruses grow as efficiently as their respective 19K-defective mutants, in1 and dl337 and pm700 and in700, in WI38 and other human cell types. Neither the accumulation of E1A cytoplasmic mRNAs nor the synthesis of E1A and other viral early proteins in these cells is altered as a result of these mutations in the 19K gene, and we conclude that the 19K protein does not play an essential role in regulating viral early gene expression or viral growth in human cells.     

Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA

The adenovirus mutant Ad2ts111 has been previously shown to contain a mutation in the early region 2A gene encoding the single-stranded-DNA-binding protein that results in thermolabile replication of virus DNA and a mutation in early region 1 that causes degradation of intracellular DNA. A recombinant virus, Ad2cyt106, has been constructed which contains the Ad2ts111 early region 1 mutation and the wild-type early region 2A gene from adenovirus 5. This virus, like its parent Ad2ts111, has two temperature-independent phenotypes; first, it has the ability to cause an enhanced and unusual cytopathic effect on the host cell (cytocidal [cyt] phenotype) and second, it induces degradation of cell DNA (DNA degradation [deg] phenotype). The mutation responsible for these phenotypes is a single point mutation in the gene encoding the adenovirus early region 1B (E1B) 19,000-molecular-weight (19K) tumor antigen. This mutation causes a change from a serine to an asparagine in the 20th amino acid from the amino terminus of the protein. Three other mutants that affect the E1B 19K protein function have been examined. The mutants Ad2lp5 and Ad5dl337 have both the cytocidal and DNA degradation phenotypes (cyt deg), whereas Ad2lp3 has only the cytocidal phenotype and does not induce degradation of cell DNA (cyt deg+). Thus, the DNA degradation is not caused by the altered cell morphology. Furthermore, the mutant Ad5dl337 does not make any detectable E1B 19K protein product, suggesting that the absence of E1B 19K protein function is responsible for the mutant phenotypes. A fully functional E1B 19K protein is not absolutely required for lytic growth of adenovirus 2 in HeLa cells, and its involvement in transformation of nonpermissive cells to morphological variants is discussed.     

Deletion of the gene encoding the adenovirus 5 early region 1b 21,000-molecular-weight polypeptide leads to degradation of viral and host cell DNA.

The adenovirus 5 mutant H5dl337 lacks 146 base pairs within early region 1B. The deletion removes a portion of the region encoding the E1B 21,000-molecular-weight (21K) polypeptide, but does not disturb the E1B-55K/17K coding region. The virus is slightly defective for growth in cultured HeLa cells, in which its final yield is reduced ca. 10-fold compared with wild-type virus. The mutant displays a striking phenotype in HeLa cells. The onset of cytopathic effect is dramatically accelerated, and both host cell and viral DNAs are extensively degraded late after infection. This defect has been described previously for a variety of adenovirus mutants and has been termed a cytocidal (cyt) phenotype. H5dl337 serves to map this defect to the loss of E1B-21K polypeptide function. In addition to its defect in the productive growth cycle, H5dl337 is unable to transform rat cells at normal efficiency.     

The efficiency of adenovirus transformation of rodent cells is inversely related to the rate of viral E1A gene expression.

While the products of the type 5 adenovirus E1A and E1B genes can initiate pathways leading to a transformed rodent cell, little is known about how the rate of viral early gene expression influences the efficiency of this process. An adenovirus mutant [E1a(r) virus] that expresses its viral E1A and E1B genes at as much as a 100-fold-reduced rate relative to wild-type virus in infected CREF or HeLa cells transforms CREF cells at an 8-fold-higher efficiency than wild-type virus. Additional studies show that the reduction in viral E1A gene expression is solely responsible for this transformation phenotype, and at this low rate of viral E1A gene expression both E1A gene products must be expressed. Unlike previously characterized viruses which transform CREF cells at frequencies greater than wild-type virus, the foci obtained following E1a(r) virus infection were indistinguishable from those arising from wild-type virus by several criteria (morphological characteristics and anchorage-independent growth). Surprisingly, an analysis of viral early gene expression from a panel of wild-type- and E1a(r) virus-transformed CREF cell lines showed similar average rates of both viral E1A and E1B gene expression. By using an adenovirus-transformed cell line that is cold-sensitive for maintenance of the transformed cell phenotype, we show that both wild-type and the E1a(r) viruses can transform these cells at equally high efficiencies at the nonpermissive temperature of 32 degrees C. Our findings suggest that the process leading to a fully transformed cell involves multiple stages, with an early stage being facilitated by a reduced rate of viral E1A gene expression.     

Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells.

Expression of the adenovirus E1A oncogene induces apoptosis which impedes both the transformation of primary rodent cells and productive adenovirus infection of human cells. Coexpression of E1A with the E1B 19,000-molecular-weight protein (19K protein) or the Bcl-2 protein, both of which have antiapoptotic activity, is necessary for efficient transformation. Induction of apoptosis by E1A in rodent cells is mediated by the p53 tumor suppressor gene, and both the E1B 19K protein and the Bcl-2 protein can overcome this p53-dependent apoptosis. The functional similarity between Bcl-2 and the E1B 19K protein suggested that they may act by similar mechanisms and that Bcl-2 may complement the requirement for E1B 19K expression during productive infection. Infection of human HeLa cells with E1B 19K loss-of-function mutant adenovirus produces apoptosis characterized by enhanced cytopathic effects (cyt phenotype) and degradation of host cell chromosomal DNA and viral DNA (deg phenotype). Failure to inhibit apoptosis results in premature host cell death, which impairs virus yield. HeLa cells express extremely low levels of p53 because of expression of human papillomavirus E6 protein. Levels of p53 were substantially increased by E1A expression during adenovirus infection. Therefore, E1A may induce apoptosis by overriding the E6-induced degradation of p53 and promoting p53 accumulation. Stable Bcl-2 overexpression in HeLa cells infected with the E1B 19K- mutant adenovirus blocked the induction of the cyt and deg phenotypes. Expression of Bcl-2 in HeLa cells also conferred resistance to apoptosis mediated by tumor necrosis factor alpha and Fas antigen, which is also an established function of the E1B 19K protein. A comparison of the amino acid sequences of Bcl-2 family members and that of the E1B 19K protein indicated that there was limited amino acid sequence homology between the central conserved domains of E1B 19K and Bcl-2. This domain of the E1B 19K protein is important in transformation and regulation of apoptosis, as determined by mutational analysis. The limited sequence homology and functional equivalency provided further evidence that the Bcl-2 and E1B 19K proteins may possess related mechanisms of action and that the E1B 19K protein may be the adenovirus equivalent of the cellular Bcl-2 protein.     

Adenovirus type 12 E1B 19-kilodalton protein is not required for oncogenic transformation in rats.

The adenovirus type 12 mutants in700 and pm700 carry site-specific mutations within the reading frame encoding the E1B 19-kilodalton protein (19K protein) which prevent the production of the intact 19K protein. In cultures of human A549 cells, these mutants grow just as well as the wild-type virus does, but they display a large-plaque (lp), cytocidal (cyt) phenotype. DNA in these infected cells is not degraded, but at late times in human KB cells infected by the mutants, the mutants display a DNA degradation (deg) phenotype. The transformation phenotype of these mutants is also host range. Although the mutants are defective for transformation of the 3Y1 rat cell line, they transform rat and mouse primary kidney cells in vitro at wild-type efficiency and are capable of inducing tumors in rats. These results support the view that the type 12 E1B 19K protein is not obligatory for oncogenic transformation.     

The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation.

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.     

cyt gene of adenoviruses 2 and 5 is an oncogene for transforming function in early region E1B and encodes the E1B 19,000-molecular-weight polypeptide

A total of 59 cytocidal (cyt) mutants were isolated from adenovirus 2 (Ad2) and Ad5. In contrast to the small plaques and adenovirus type of cytopathic effects produced by wild-type cyt+ viruses, the cyt mutants produced large plaques, and the cytopathic effect was characterized by marked cellular destruction. cyt mutants were transformation defective in established rat 3Y1 cells. cyt+ revertants and cyt+ intragenic recombinants recovered fully the transforming ability of wild-type viruses. Thus, the cyt gene is an oncogene responsible for the transforming function of Ad2 and Ad5. Genetic mapping in which we used three Ad5 deletion mutants (dl312, dl313, and dl314) as reference deletions located the cyt gene between the 3' ends of the dl314 deletion (nucleotide 1,679) and the dl313 deletion (nucleotide 3,625) in region E1B. Restriction endonuclease mapping of these recombinants suggested that the cyt gene encodes the region E1B 19,000-molecular-weight (175R) polypeptide (nucleotides 1,711 to 2,236). This was confirmed by DNA sequencing of eight different cyt mutants. One of these mutants has a single missense mutant, two mutants have double missense mutations, and five mutants have nonsense mutations. Except for one mutant, these point mutations are not located in any other known region E1B gene. We conclude that the cyt gene codes for the E1B 19,000-molecular-weight (175R) polypeptide, that this polypeptide is required for morphological transformation of rat 3Y1 cells, and that simple amino acid substitutions in the protein can be sufficient to produce the cyt phenotype.     

p53 Status Does Not Determine Outcome of E1B 55-Kilodalton Mutant Adenovirus Lytic Infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G₁. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39°C, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39°C. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.     

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.     

p300 binding by E1A cosegregates with p53 induction but is dispensable for apoptosis.

E1A expression during adenovirus infection induces apoptosis. E1A expression causes accumulation of the p53 tumor suppressor protein, and E1A-induced apoptosis is p53 mediated in primary rodent cells, implying that p53 induction may be linked to apoptosis induction by E1A. Adenoviruses containing mutations in the E1A gene were tested for the ability to trigger both p53 accumulation and the appearance of enhanced cytopathy (cyt phenotype) and degradation of DNA (deg phenotype), indicative of apoptosis in infected HeLa cells. The adenoviruses had mutations which disrupted the pRb- and/or p300-binding activities of E1A so that the relationship between p53 induction and apoptosis and binding to these cellular proteins by E1A could be determined. An E1A mutation that specifically disrupted the p300-binding activity failed to induce p53 accumulation, whereas mutations in E1A which affected pRb binding induced p53 accumulation. Thus, p300 binding was required and pRb binding was dispensable for E1A-mediated accumulation of p53 in HeLa cells. All the E1A mutant viruses, regardless of the ability to induce p53 accumulation, induced the cyt and deg phenotypes, suggesting that p53 induction in infected HeLa cells was not essential for apoptosis, nor was binding of E1A to the pRb and/or p300 protein. The possibility that E1A induced a p53-independent apoptosis pathway was tested by analyzing the appearance of the cyt and deg phenotypes in Saos-2 cells, which were null for both alleles of p53, upon adenovirus infection. An adenovirus expressing wild-type 12S E1A induced both the cyt and deg phenotypes in Saos-2 cells, as did all the E1A mutant viruses. Thus, E1A expression during infection of human cells may trigger redundant p53-independent and -dependent apoptotic pathways.     

Adenovirus 12 nononcogenic mutants: oncogenicity of transformed cells and viral proteins synthesized in vivo and in vitro

Several nononcogenic cyt mutants of adenovirus type 12 induced the same E1A (55,000 [55K] and 25K) and E1B polypeptides (55K, 19K, and 17K) as did the wild-type virus, except that cyt 68 did not induce the E1B 19K protein. Tumorigenicity tests showed cells transformed by cyt 68 to be highly oncogenic in vivo. Therefore, it was concluded that the E1B 19K polypeptide is not necessary for tumor induction but may be involved in the efficiency of transformation.     

Overexpression of the E1B 55-kilodalton (482R) protein of human adenovirus type 12 appears to permit efficient transformation of primary baby rat kidney cells in the absence of the E1B 19-kilodalton protein.

To analyze the structure and function of the E1B 19,000-molecular-weight protein (19K protein) (163R) of human adenovirus type 12, mutants were produced at various positions across the 163R-coding sequence. Viruses bearing mutations within the first 100 or so amino acids yielded unstable 163R-related products, induced DNA degradation and enhanced cytopathic effect (cyt/deg phenotype) in KB cells, and transformed primary rodent cells at much lower efficiencies than wild-type (wt) virus. Deletion of the final 16 residues at the carboxy terminus had no phenotypic effect. Alteration of residue 105 reduced transforming efficiency significantly, suggesting that this region of 163R is functionally important. Disruption of the AUG initiation codon at nucleotide 1542 blocked production of 163R completely but resulted in higher levels of E1B 55K-482R protein synthesis and a transforming efficiency similar to that of wt virus. These data suggested that while 163R is of some importance, normal transforming efficiencies can be obtained in its absence if 482R is overexpressed.     

Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.     

Transformation of rodent cells by DNA extracted from transformation-defective adenovirus mutants

Complementation group II host range mutants of adenovirus type 5 which map in early region 1B (E1B, 4.5 to 11.0 map units) have been shown to be defective for the synthesis of the E1B 58,000-dalton (58K) antigen in infections of HeLa or KB cells (Lassam et al., Cell 18:781-791, 1979) and unable to transform cultured rodent cells (Graham et al., Virology 86:10-21, 1978). In this report we show that DNA extracted from group II mutants hr6 and hr50 can transform rat cells with the same efficiency as wild-type DNA. Furthermore, group II mutant-transformed hamster cells were shown to contain no detectable E1B 58K tumor antigen but were capable of inducing tumors in newborn hamsters. Hamster cell lines 1019-3 and 1019-C3, transformed by hr50 DNA, produced no detectable quantities of either the E1B 58K or 19K antigen but nonetheless exhibited a fully transformed oncogenic phenotype. Our results show that the E1B 58K antigen is not absolutely required for oncogenic transformation and suggest that even cells lacking the 19K protein can be oncogenic.