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.     

Mapping of an adenovirus function involved in the inhibition of DNA degradation.

A function involved in the inhibition of DNA degradation has been assigned through complementation tests to a product of region E1b of the adenovirus genome (between 4.5 and 10.5 map units). DNA degradation induced by the adenovirus type 12 (Ad12) cyt mutant H12cyt70 and the Ad5 early deletion mutant dl313 (with the deletion between 3.5 and 10.7 map units) was inhibited by coinfection with Ad5 region E1a (between 0 and 4.5 map units) mutants dl312 and hr1 and region E1b mutant hr6. The defect of inhibition of DNA degradation in Ad5 dl313 was also complemented in 293 cells. This DNase-inhibitory function does not appear to involve polypeptide IX or the 58,000-dalton polypeptide. Wild-type Ad12 induced DNA degradation in hamster embryo cells, suggesting that the DNase-inhibitory function is not expressed in these nonpermissive cells. Additional evidence suggests the involvement of a second viral product which positively influences the DNase activity and which appears to be an early function.     

Isolation of transformation-defective, replication-nondefective early region 1B mutants of adenovirus 12.

We isolated three adenovirus 12 early region 1B mutants (in205B, in205C, and dl205) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. These mutants could replicate efficiently in human embryo kidney or KB cells but showed markedly reduced transforming capacities both in vitro and in vivo. In cells infected with the mutants, the early region 1B gene was transcribed efficiently. In cells infected with in205B, the products corresponding to the early region 1B-coded 19,000-molecular-weight polypeptide was detected by in vitro translation but not immunoprecipitated extract of labeled cells. In cells infected with in205C or dl205, the products corresponding to the same polypeptide were not detected by either in vitro translation or immunoprecipitation of labeled cell extracts. The results suggest that the 19,000-molecular-weight polypeptide encoded by early region 1B is required for cell transformation but not for viral propagation.     

Characterization of Marek's disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames.

We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome. A deletion mutant (GA delta 4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli. This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorf1, Sorf2, and Sorf3. Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US2lac insertion mutant (J. L. Cantello, A. S. Anderson, A. Francesconi, and R. W. Morgan, J. Virol. 65:1584-1588, 1991). The isolation of these mutants indicates that the relevant genes are not required for growth of MDV in chicken embryo fibroblasts. The mutants had early growth kinetics indistinguishable from those of their parent viruses; however, 5 to 7 days after being plated, the US1 insertion mutant (US1lac) and the GA delta 4.8lac deletion mutant showed a 5- to 10-fold decrease in virus growth. This decrease in virus accumulation correlated with a 30 to 50% decrease in plaquing efficiency when these viruses were plated onto established versus fresh chicken embryo fibroblast monolayers compared with a 10 to 15% decrease seen for the parent viruses and for the US10lac or US2lac insertion mutants. Finally, GA delta 4.8lac could be reisolated from chickens, indicating that the deleted genes are not required for the infection of chickens following intra-abdominal inoculation of an attenuated serotype 1 MDV.     

Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus.

The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated. The three major proteins are phosphorylated but not glycosylated and share antigenic determinants recognized by monoclonal antibodies. The 84 and 76-kd polypeptides represent post-translational modification products of the 89-kd protein. Accordingly, in vitro translation of IE infected cell RNA revealed only the 89-kd polypeptide. The viral origin of the RNA species directing the synthesis of the major 89-kd IE polypeptide was verified by hybrid selection of IE RNA with DNA fragments representing the region from 0.769 to 0.815 map units of the murine cytomegalovirus genome. IE polypeptides were found to be located in the nuclei and the cytoplasm of infected cells. Studies on the kinetics of IE polypeptide synthesis revealed negative regulatory effects on IE gene expression correlated with the synthesis of early proteins.     

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.     

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells.

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.     

Role of small t antigen in the acute transforming activity of SV40

A plasmid, pHR402, containing SV40 sequences that include a truncated early region bearing an intact t-coding sequence and a functionally intact late region, was introduced into thymidine kinase deficient (tk-) mouse L cells by cotransformation with a cloned tk gene. tk+ cotransformants synthesized SV40 t but not T antigen, and no truncated T-coding sequence products were detected. The viral sequences of pHR402 were reconstituted as a virus in COS1 cells, and acute infection of untransformed mouse cells with this viral stock (SV402) also led to the appearance of t but not T or a truncated T. Abortive transformation assays of such infected cells were negative, as were those performed on the same cells infected with either of two viral mutants (dl883 and dl884), each of which leads to T but not t synthesis. However, mixed infection with SV402 and either dl883 or dl884 led to a clear abortive and permanent transformation response. Thus, at least in part, t and T appear to function in a complementary fashion in eliciting transformation expression by SV40-infected cells.     

trans-dominant interference of type 5 adenovirus E1a mutants in cell transformation.

Two type 5 adenovirus (Ad5) early region 1a (E1a) mutants, H5in104 and H5dl105, were impaired in viral replication and cell transformation. In addition, these mutants trans dominantly inhibited the frequency with which H5sub309, a phenotypically wild-type mutant, and H5dl520, a high-frequency transformation mutant, transformed CREF cells. Inhibition of transformation varied in proportion to the input ratio of mutant to coinfecting virus. It was found that H5in104, but not H5dl105, could not complement Ad5 E1b mutants that failed to synthesize 19- or 55-kDa E1b product. H5dl105 yielded 10-fold less virus than the wild-type did in 293 cells, which constitutively express E1a and E1b products; similar low yields were also observed with H5in104 and H5dl105 in another E1a- and E1b-expressing transformed cell line, KB16. Marker rescue and DNA sequence analyses, however, indicated that the phenotypes of H5in104 and H5dl105 were the result of their respective E1a mutations. The data presented are the first to demonstrate that mutants of animal viruses can effect dominant interference with the viral function(s) that produce cell transformation.     

Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells.

A series of adenovirus type 5 (Ad5) deletion, insertion and substitution mutants, some of which are defective for transformation of rat cells, have been isolated. The mutants were selected as variants which lack the Xba I endonuclease cleavage site at 4 map units on the viral chromosome. The deletions range in size from 150-2300 bp and are located between 1.5 and 10.5 map units. The mutants can be propagated in 293 cells (Ad5-transformed human embryonic kidney cells), but are defective for growth in HeLa or human embryonic kidney cells. No viral DNA synthesis was observed in mutant virus-infected HeLa cells. All but one of the deletion mutants tested were defective for transformation of rat embryo and rat embryo brain cells.     

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.     

Location and identification of the genes for adenovirus type 2 early polypeptides

Virus-specific RNA was prepared from cells early after adenovirus type 2 infection and fractionated by hybridization to specific fragments of viral DNA. The viral mRNA was used to program cell-free protein synthesis, and the products were analyzed by electrophoresis. The genes for the early polypeptides of apparent molecular weight 44,000, 15,000, 72,000, 15,500, 19,000, and 11,000 daltons were located, respectively, between positions 0–4.1, 4.1–16.7, 58.5–70.7, 75.9–83.4, 89.7–98.6, and 89.7–98.6 of the conventional adenovirus DNA map. The polypeptide of molecular weight 72,000 daltons was shown to be the single-strand DNA-binding protein described by others. RNAs from three different adeno-transformed cell lines each program the synthesis in vitro of predominantly the 15K polypeptide, as well as variable amounts of the polypeptide of molecular weight 44,000 daltons. The genes for these two polypeptides are located in the portion of DNA known to be required for transformation of rodent cells by adenovirus.     

Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts.

The simian virus 40 large T antigen immortalizes growing primary cells in culture. In addition, this viral oncoprotein cooperates with an activated ras protein to produce dense foci on monolayers of rat embryo fibroblasts (REF). The relationship between independent immortalization and cooperative transformation with ras has not been defined. Previously, two regions of T antigen were shown to contain immortalization activities. An N-terminal fragment consisting of amino acids 1 to 147 immortalizes rodent cells (L. Sompayrac and K. J. Danna, Virology 181:412-415, 1991). Loss-of-function analysis indicated that immortalization depended on integrity of the T-antigen segments containing amino acids 351 to 450 and 533 to 626 (T. D. Kierstead and M. J. Tevethia, J. Virol. 67:1817-1829, 1993). The experiments described here were directed toward determining whether these same T-antigen regions were sufficient for cooperation with ras. Initially, constructs that produce T antigens containing amino acids 176 to 708 (T176-708) or 1 to 147 were tested in a ras cooperation assay. Both polypeptides cooperated with ras to produce dense foci on monolayers of primary REF. These results showed that T antigen contains two separate ras cooperation activities. In order to determine the N-terminal limit of the ras cooperation activity contained within the T176-708 polypeptide, a series of constructs designed to produce fusion proteins containing T-antigen segments beginning at residues 251, 301, 337, 351, 371, 401, 451, 501, 551, 601, and 651 was generated. Each of these constructs was tested for the capacity to cooperate with ras to produce dense foci on REF monolayers. The results indicated that a polypeptide containing T-antigen amino acids 251 to 708 (T251-708) was sufficient to cooperate with ras, whereas the more extensively truncated products were not. The abilities of the N-terminally truncated T antigens to bind p53 were examined in p53-deficient cells infected with a recombinant vaccinia virus expressing a phenotypically wild-type mouse p53. The results showed that polypeptides containing T-antigen amino acids 251 to 708, 301 to 708, 337 to 708, or 351 to 708 retained p53-binding capacity. The introduction into the T251-708 polypeptide of deletions that either prevented p53 binding (dl434-444) or did not prevent p53 binding (dl400) abrogated ras cooperation. These results indicated that although p53 binding may be necessary for ras cooperation, an additional, as-yet-undefined activity contained within the T251-708 polypeptide is needed.     

Site-directed mutagenesis of the simian virus 40 large T-antigen gene: replication-defective amino acid substitution mutants that retain the ability to induce morphological transformation.

We used a heteroduplex deletion loop mutagenesis procedure for directing sodium bisulfite-induced mutations to specific sites on viral or plasmid DNA to generate a series of SV40 large T-antigen point mutants. The mutations were directed to a region of the T-antigen gene, 0.5 map units, that is thought to be important for interaction of the protein with the viral origin of DNA replication. Of the 16 mutants reported here, 10 had lost the ability to replicate their DNA, and 3 others showed a reduced level of replication compared to wild type. All of the mutants tested were capable of transforming rat cells in culture by the dense focus assay. We conclude that the sequences of the early region around 0.5 map units are critical for the replication of viral DNA but not for the transformation function of T antigen.     

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.     

The 55K protein on the 5'' termini of adenovirus type 2 DNA is unrelated to virus-coded candidate transformation proteins (E1-53K, E1-40K-50K) and DNA-binding proteins (E2-42K/47K/73K).

A polypeptide of 55,000 daltons (55K) is linked, probably covalently, to the K' termini of adenovirus type 2 DNA. The 55K polypeptide is synthesized during early stages of infection (T. Yamashita, M. Arens, and M. Green, J. Virol. 30: 497-507, 1979) and thus may function in viral DNA replication, gene regulation, or cell transformation. Several virus-coded early polypeptides have been identified that could correspond to the terminal 55K, including the E1-40K-50K and E1-53K candidate transformation polypeptides and the E2-42K/47K/73K single-stranded DNA-binding polypeptide. We show here that two-dimensional tryptic [35S]methionine-peptide maps of the terminal 55K differ completely from [35S]methionine-peptide maps of four related E1-40K-50K polypeptides, the E1-53K, and the related E2-42K, E2-47K, and E2-73K polypeptides. We conclude that the terminal 55K polypeptide does not correspond to any of the known virus-coded early polypeptides.