The E1B 19-kilodalton protein is not essential for transformation of rodent cells in vitro by adenovirus type 5.

The newly constructed adenovirus type 5 mutant in1 carries a single AT base pair insertion immediately after nucleotide position 1715 in the E1B gene sequence which destroys the proximal AUG normally present in E1B messages and prevents production of intact E1B 19-kDa protein in infected cells. We have used in1, variants of in1 containing mutant alleles of viral genes known to enhance transformation frequency, and adenovirus type 5 mutant dl337 (S. Pilder, J. Logan, and T. Shenk, J. Virol. 52:664-671, 1984), in which the sequence between nucleotides 1770 and 1916 within the 19-kDa reading frame is deleted, to test the generally accepted hypothesis that this E1B protein is essential for the transformation of rodent cells and maintenance of the transformed phenotype. We find that these mutants transform rat embryo cells, rat kidney and mouse kidney primary cells, and cells of the 3Y1 rat line with decreased frequencies only when virus is added to these various cells at high input multiplicities of infection. In contrast, when lower doses of virus are used, the mutants transform with wild-type frequencies. Cells infected with higher doses of mutant virus show increased levels of DNA degradation and cell killing compared with those of cells infected with the same levels of wild-type virus, and these effects most likely contribute to the decreased transformation frequencies observed. On the basis of these results and the results of phenotypic analyses of numerous transformants, we propose that the E1B 19-kDa protein is not required for induction and/or maintenance of transformed-cell characteristics in rodent cells infected with adenovirus type 5.     

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.     

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.     

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 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.     

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.     

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.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

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.     

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.     

Adenovirus cyt+ locus, which controls cell transformation and tumorigenicity, is an allele of lp+ locus, which codes for a 19-kilodalton tumor antigen.

The early region E1b of adenovirus type 2 (Ad2) codes for two major tumor antigens of 53 and 19 kilodaltons (kd). The adenovirus lp+ locus maps within the 19-kd tumor antigen-coding region (G. Chinnadurai, Cell 33:759-766, 1983). We have now constructed a large-plaque deletion mutant (dl250) of Ad2 that has a specific lesion in the 19-kd tumor antigen-coding region. In contrast to most other Ad2 lp mutants (G. Chinnadurai, Cell 33:759-766, 1983), mutant dl250 is cytocidal (cyt) on infected KB cells, causing extensive cellular destruction. Cells infected with Ad2 wt or most of these other Ad2 lp mutants are rounded and aggregated without cell lysis (cyt+). The cyt phenotype of dl250 resembles the cyt mutants of highly oncogenic Ad12, isolated by Takemori et al. (Virology 36:575-586, 1968). By intertypic complementation analysis, we showed that the Ad12 cyt mutants indeed map within the 19-kd tumor antigen-coding region. The transforming potential of dl250 was assayed on an established rat embryo fibroblast cell line, CREF, and on primary rat embryo fibroblasts and baby rat kidney cells. On all these cells, dl250 induced transformation at greatly reduced frequency compared with wt. The cells transformed by this mutant are defective in anchorage-independent growth on soft agar. Our results suggest that the 19-kd tumor antigen (in conjunction with E1a tumor antigens) may play an important role in the maintenance of cell transformation. Since we have mapped the low-oncogenic or nononcogenic Ad12 cyt mutants within the 19-kd tumor antigen-coding region, our results further indicate that the 19-kd tumor antigen also directly or indirectly plays an important role in tumorigenesis of Ad12. Our results show that the cyt+ locus is an allele of the lp+ locus and that the cyt phenotype may be the result of mutations in specific domains of the 19-kd tumor antigen.     

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.     

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.     

Activated H-ras rescues E1A-induced apoptosis and cooperates with E1A to overcome p53-dependent growth arrest.

The adenovirus E1A oncogene products stimulate DNA synthesis and cell proliferation but fail to transform primary baby rat kidney (BRK) cells because of the induction of p53-mediated programmed cell death (apoptosis). Overexpression of dominant mutant p53 (to abrogate wild-type p53 function) or introduction of apoptosis inhibitors, such as adenovirus E1B 19K or Bcl-2 oncoproteins, prevents E1A-induced apoptosis and permits transformation of BRK cells. The ability of activated Harvey-ras (H-ras) to cooperate with E1A to transform BRK cells suggests that H-ras is capable of overcoming the E1A-induced, p53-dependent apoptosis. We demonstrate here that activated H-ras was capable of suppressing apoptosis induced by E1A and wild-type p53. However, unlike Bcl-2 and the E1B 19K proteins, which completely block apoptosis but not p53-dependent growth arrest, H-ras expression permitted DNA synthesis and cell proliferation in the presence of high levels of wild-type p53. The mechanism by which H-ras regulates apoptosis and cell cycle progression is thereby strikingly different from that of the E1B 19K and Bcl-2 proteins. BRK cells transformed with H-ras and the temperature sensitive murine mutant p53(val 135), which lack E1A, underwent growth arrest at the permissive temperature for wild-type p53. p53-dependent growth arrest, however, could be relieved by E1A expression. Thus, H-ras alone was insufficient and cooperation of H-ras and E1A was required to override growth suppression by p53. Our data further suggest that two complementary growth signals from E1A plus H-ras can rescue cell death and thus permit transformation.     

Cellular alterations dependent upon the polyoma virus Hr-t function: separation of mitogenic from transforming capacities.

Hr-t mutants of polyoma virus are restricted in their growth properties (host range) and defective in cell transformation and tumor induction. The present study indicates that these mutants have lost the ability to induce morphological transformation, but have retained a mitogenic function. Thus an early and dramatic difference between wild-type virus and hr-t mutant-infected cultures of rat fibroblasts is the morphological change in individual cells observed by light, fluorescence and scanning electron microscopy. Viruses containing an intact hr-t function (wild-type virus and ts-a mutants) induce a transformed phenotype consisting of stellate cell shape, loss of defined cytoplasmic actin architecture, cellular "underlapping," and increased nuclear and nucleolar sizes. These prominent alterations constitute an abortive transformation, peaking 24-48 hr post-infection, and subsequently resolving in most or all of the cells. In contrast, cells infected with hr-t mutants do not develop the above structural changes, but rather retain their preinfection appearance. Both wild-type virus and hr-t mutants induce cellular DNA synthesis in confluent monolayers of rat cells beginning 12-14 hr post-infection. Flow microfluorometric (FMF) analysis confirms the viral mediated transit of cells from the G1 to the S and G2 phases of the cell cycle, as well as an increase in the proportion of cells with an 8N (octaploid) DNA content. Approximately 50% of the clones isolated from wild-type-infected cultures are polyploid. Stable transformants are found among these polyploid clones, but the majority of the latter resemble the parental cells in their morphology and growth properties. Polyploid clones are derived from hr-t mutant-infected cultures at a much lower frequency, similar to that of mock-infected cultures. Data obtained by sequential labeling of infected cultures with 3 H-thymidine and 5-bromo-deoxyuridine, together with cell number quantitation, indicate that hr-t mutants promote only a single round of cell division, while the wild-type virus and ts-a mutants promote multiple rounds. Loss of the hr-t function in polyoma virus therefore reveals a residual viral mitogenic activity, but prevents the virus from effecting morphological transformation of cells with concomitant loss of defined actin cables, polyploidization and multiple cycles of cell division in confluent cultures.