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.     

Dependence of tumor-forming capacities of cells transformed by recombinants between adenovirus types 5 and 12 on expression of early region 1.

Recombinants between an adenovirus type 5 (Ad5) deletion mutant and the Ad12 DNA fragment containing early region 1 (E1) were isolated from cells cotransfected with the EcoRI-C fragment of Ad12 DNA and Ad5 dl312 (deletion in E1A) DNA (rcA) and from cells cotransfected with the SalI-C fragment of Ad12 DNA and Ad5 dl312 DNA (rcB). No recombinant was isolated from cells cotransfected with Ad5 dl313 (deletion in E1B) DNA and restriction fragments of Ad12 DNA. Both rcA and rcB are defective and able to replicate in human embryo kidney (HEK) and KB cells with complementation by dl312. Both rcA and rcB formed Ad12 T antigen g, but not T antigen f, in infected HEK and KB cells. In rcA- and rcB-infected cells, Ad5 E1B and Ad12 E1A genes are transcribed. Heteroduplex and size analyses of rcA-1 or rcB-1 DNA fragments hybridized with Ad12 DNA revealed that rcA-1 DNA has a deletion between 5 and 15 map units with an insertion of a portion of Ad12 DNA (10%) and that rcB-1 DNA has a deletion between 70 and 80 map units with an insertion of a portion of Ad12 DNA (10%). The transformed cell lines, RCAY and RCBY, were established after infection of rat 3Y1 cells with rcA and rcB, respectively. Both Ad5 and Ad12 DNA sequences are contained in these cells. In RCAY cells, Ad12 T antigen g is detected, but Ad12 T antigen f is not. In RCBY cells, both Ad12 T antigen g and f are detected. Only the Ad12 E1A gene is transcribed in RCAY cells, whereas Ad5 E1B, Ad12 E1A, and Ad12 E1B genes are transcribed in RCBY cells. In soft-agar cultures, RCBY cells form large colonies, whereas RCAY cells form only tiny colonies. RCBY cells form tumors as efficiently as 12WY cells in transplanted rats. RCAY cells formed tumors inefficiently. Ad5-transformed 5WY cells do not form tumors. These observations indicate that the efficient tumor formation by RCBY cells is dependent on the expression of the Ad12 E1A and E1B genes, whereas the inefficient tumor formation by RCAY cells is due to the expression of only the Ad12 E1A gene.     

Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.     

Transformation of rat cells by cyt mutants of adenovirus type 12 and mutants of adenovirus type 5.

Several mutants with much reduced oncogenicity (spontaneous mutants H12 cyt 52 and H12 cyt 70 and UV-induced mutants H12 cyt 61, H12 cyt 62, and H12 cyt 68) of the highly oncogenic adenovirus type 12 (Ad12) were studied for their ability to transform primary baby rat kidney cells. Four of the mutants showed much reduced capacity to transform cells in vitro, while H12 cyt 61 transformed cells as efficiently as the wild-type virus. Viral gene expression in several cell lines established from cultures infected by cyt mutants was studied, and it was found that viral sequences belonging to the left 16% of Ad12 were always transcribed. These results suggest that the function of the transformed state is not defective in the cyt mutants studied. Heterotypic complementation studies showed that the defect(s) in a cyt mutant can be corrected by an Ad7 function. Ad5 dl 313, with a deletion between 3.5 and 10.5 map units, transformed rat cells only at high multiplicity. These results suggest that the region E1B of adenoviruses may be required for efficient transformation of rat cells.     

Hybrids of Human and Monkey Adenoviruses (Adeno-Adeno Hybrids) That Can Reproduce in Monkey Cells: Biological and Molecular Genetic Peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.     

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.     

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 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.     

Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells.

In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed 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.     

Adenovirus E1A gene induction of susceptibility to lysis by natural killer cells and activated macrophages in infected rodent cells.

Rodent cells immortalized by the E1A gene of nononcogenic adenoviruses are susceptible to lysis by natural killer (NK) cells and activated macrophages. This cytolysis-susceptible phenotype may contribute to the rejection of adenovirus-transformed cells by immunocompetent animals. Such increased cytolytic susceptibility has also been observed with infected rodent cells. This infection model provided a means to study the role of E1A gene products in induction of cytolytic susceptibility without cell selection during transformation. Deletion mutations outside of the E1A gene had no effect on adenovirus type 2 (Ad2) or Ad5 induction of cytolytic susceptibility in infected hamster cells, while E1A-minus mutant viruses could not induce this phenotype. E1A mutant viruses that induced expression of either E1A 12S or 13S mRNA in infected cells were competent to induce cytolytic susceptibility. Furthermore, there was a correlation between the accumulation of E1A gene products in Ad5-infected cells and the level of susceptibility of such target cells to lysis by NK cells. The results of coinfection studies indicated that the E1A gene products of highly oncogenic Ad12 could not complement the lack of induction of cytolytic susceptibility by E1A-minus Ad5 virus in infected cells and also could not block induction of this infected-cell phenotype by Ad5. These data suggest that expression of the E1A gene of nononcogenic adenoviruses may cause the elimination of infected cells by the immunologically nonspecific host inflammatory cell response prior to cellular transformation. The lack of induction of this cytolysis-susceptible phenotype by Ad12 E1A may result in an increased persistence of Ad12-infected cells in vivo and may lead to an increased Ad12-transformed cell burden for the host.     

Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus.

The growth of adeno-associated virus (AAV) is dependent upon helper functions provided by adenovirus. We investigated the role of adenovirus early gene region 1 in the AAV helper function by using six adenovirus type 5 (Ad5) host range mutants having deletions in early region 1. These mutants do not grow in human KB cells but are complemented by and grow in a line of adenovirus-transformed human embryonic kidney cells (293 cells); 293 cells contain and express the Ad5 early region 1 genes. Mutants having extensive deletions of adenovirus early region 1a (dl312) or regions 1a and 1b (dl313) helped AAV as efficiently as wild-type adenovirus in 293 cells, but neither mutant helped in KB cells. No AAV DNA, RNA, or protein synthesis was detected in KB cells in the presence of the mutant adenoviruses. Quantitative blotting experiments showed that at 20 h after infection with AAV and either dl312 or dl313 there was less than one AAV genome per cell. In KB cells infected with AAV alone, the unreplicated AAV genomes were detected readily. Apparently, infection with adenovirus mutant dl312 or dl313 results in degradation of most of the infecting AAV genomes. We suggest that at least an adenovirus region 1b product (and perhaps a region 1a product also) is required for AAV DNA replication. This putative region 1b function appears to protect AAV DNA from degradation by an adenovirus-induced DNase. We also tested additional Ad5 mutants (dl311, dl314, sub315, and sub316). All of these mutants were inefficient helpers, and they showed varying degrees of multiplicity leakiness. dl312 and dl313 complemented each other for the AAV helper function, and each was complemented by Ad5ts125 at the nonpermissive temperature. The defect in region 1 mutants for AAV helper function acts at a different stage of the AAV growth cycle than the defect in the region 2 mutant ts125.     

The relationship between region E1a and E1b of human adenoviruses in cell transformation

Baby rat kidney (BRK) cells were transfected either with intact region E1 DNA of adenovirus type 5 (Ad5) or with mixtures of DNA fragments containing the separated E1a and E1b regions. The results showed that mixtures of regions E1a and E1b transform with a similar efficiency as intact region E1. DNA fragments containing region E1b alone have no detectable transforming activity in primary BRK cells nor in established rat cell lines. When region E1a and Ad5 was combined with region E1b and Ad12 complete transformation was also obtained. Characterization of the cell lines transformed by separated E1a and E1b regions have led to the following conclusions: (1) Expression of region E1b is not dependent on specific linkage to region E1a as it occurs in the intact E1 region. (2) Region E1b is normally expressed into the corresponding major adenovirus T antigens (65,000 and 19,000 Mr with region E1b of Ad5; 60,000 and 19,000 Mr with E1b or AD12). (3) Region E1b of Ad12 can be activated by region E1a of Ad5 indicating that the Ela regions of both serotypes are functionally similar in transformation. (4) Cell lines containing region E1b of Ad5 are weakly oncogenic in nude mice whereas cells containing E1b of Ad12 are highly oncogenic in nude mice, indicating that the degree of oncogenicity is determined by region E1b.     

Production of a monospecific antiserum against the early region 1A proteins of adenovirus 12 and adenovirus 5 by an adenovirus 12 early region 1A-beta-galactosidase fusion protein antigen expressed in bacteria.

Antisera were prepared against the amino acid sequences encoded within the N-terminal half of the adenovirus 12 (Ad12) early region 1A (E1A) gene. This was accomplished by construction of a plasmid vector which encoded the N-terminal 131 amino acids of Ad12 E1A joined in frame to the coding sequence of beta-galactosidase. After induced synthesis in Escherichia coli, the Ad12 E1A-beta-galactosidase fusion protein (12-1A-FP) was extracted with urea and used to raise antibodies in rabbits. The 12-1A-FP antisera immunoprecipitated major phosphoproteins of 39,000 and 37,000 apparent molecular weights from Ad12-transformed and infected cells. The 12-1A-FP antisera also immunoprecipitated E1A phosphoproteins from Ad5-transformed and infected cells. Immunospecificity of the 12-1A-FP antisera was demonstrated by the ability of 12-1A-FP antigen to block immunoprecipitation of E1A proteins. Furthermore, E1A proteins immunoprecipitated from in vivo-labeled cells comigrated with those translated in vitro by RNA that had been hybridization selected to E1A DNA.     

Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene.

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.