Adenovirus transcription: III. Mapping of viral RNA sequences in cells productively infected by adenovirus type 5

Separated strands of restriction endonuclease Eco R1 and Hpa 1 fragments of ³²P-labeled Adenovirus 5 DNA have been used in saturation hybridization experiments with cytoplasmic RNA extracted from human cells infected with Adenovirus type 5. The results of such experiments have allowed the construction of maps of the regions of the viral genome complementary to both “early” and “late” Adenovirus 5 RNA. At early times during the lytic cycle, about 25% of the viral genome is expressed as mRNA. Adenovirus 5 DNA sequences complementary to “early” mRNA comprise four discrete regions, two on each strand, of the viral genome: except in one instance, these correspond to the regions of the Adenovirus 2 genome complementary to “early” Adenovirus 2 mRNA. In the exceptional case, “early” Adenovirus 5 mRNA appears to contain not only sequences corresponding, at least in position, to those expressed in Adenovirus 2-infected cells, but also some additional, adjacent sequences, reminiscent of the situation in human cells infected with the Adenovirus 2/Simian virus 40 hybrid viruses, Ad2⁺ND₁ and Ad2⁺ND₃ (Flint et al., 1975a).All, or almost all, the information encoded by the Adenovirus 5 genome is expressed as mRNA during the lytic cycle and most of the exclusively “late” mRNA is complementary to the r strand of Adenovirus 5 DNA. The separated strands of fragments of ³²P-labeled Adenovirus 2 DNA generated by the restriction endonuclease Bam Hl have been used to improve the resolution of maps of both Adenovirus 2 and Adenovirus 5 “late” mRNA, which are described here.     

Tumorigenicity and viral gene expression in rat cells transformed by Ad 12 virions or by the EcoRI c fragment of Ad 12 DNA

Several rat kidney cell lines transformed either by Ad 12 virions or by the EcoRI C fragment (left 16%) of Ad 12 DNA have been characterized with respect to viral DNA content, viral gene expression, and tumorigenicity. Virion transformed cells contained viral DNA sequences from most regions of the viral genome while EcoRI C fragment transformed cells contained only part of the C fragment. All cell lines transcribed DNA sequences from the left 6.7% (HindIII F fragment) of the genome while some lines transcribed sequences extending into the adjacent HindIII H fragment. Virus transformed and DNA transformed lines induced tumors in immunocompetent rats with equal efficiency.     

Characterization of tumor antigens in cells transformed by fragments of adenovirus type 5 DNA

The Ad5 T antigens in a number of transformed cell lines were isolated by immunoprecipitation with sera from hamsters bearing tumors induced by cells containing only Ad5 early region 1 (E1) DNA. The proteins were characterized by SDS-polyacrylamide gel electrophoresis. Major T antigens with molecular weights of 65K and 19K could be identified in cells transformed by virions or intact viral DNA. Minor species of 52K, 45K, 42-34K, 25K, 18K, 17K, 16K, and 15K were also found in variable amounts. Only a very limited number of anti-T sera from individual hamsters were able to precipitate all of these proteins; the majority precipitated the larger T antigens only. In particular, the 19K protein seemed weakly antigenic. Ad5 T antigens in primary rat kidney cells transformed with the following Ad5 DNA fragments were also analyzed: XhoI-C, BglII-D, HindIII-G, KpnI-H, and HpaI-E (comprising the left-most 15, 9.5, 8, 5.9, and 4.5% of the genome, respectively). The number of T antigens decreased with the size of the DNA fragment used for transformation. Cells transformed by the 15% fragment were similar to normal transformed cells, while all other cells lacked the major 65K T antigen. In addition, cells transformed by the two smallest fragments lacked the 19K species. All fragment-transformed cells contained small amounts of T antigens in the 32–52K size class, indicating that these proteins are encoded (or induced) by the smallest transforming fragment of 4.5%. Evidence is presented that the 19K protein is responsible for the induction of a number of transformed properties.     

Mechanism of infection by Epstein-Barr virus. II. Comparison of viral DNA from HR-1 and superinfected Raji cells by restriction enzymes.

Virus DNA (RS virus DNA) was directly isolated from Raji cells superinfected with Epstein-Barr virus derived from P3HR-1 cells and compared with original superinfecting virus DNA from P3HR-1 cells (HR-1 virus DNA) in agarose-gel electrophoresis after digestion with various restriction enzymes. EcoR-1 digestion of RS virus DNA produced 15 fragments identical to those from HR-1 virus DNA. However, two fragments, EcoR1 No. 6 (10 × 10⁶ daltons) and EcoR1 No. 11 (4.6 × 10⁶ daltons), observed in HR-1 virus DNA were not detected in RS virus DNA from superinfected Raji cells. In addition, the EcoR1 No. 4 (13.5 × 10⁶ daltons) fragment of RS virus DNA showed a molar ratio of 2 whereas HR-1 virus DNA produced the same fragment with a molar ratio of 1. Electrophoresis patterns of virus DNA digested with Hind III, Bam H-I, Hpa I, and Sal I were also examined. In general, both types of virus DNA produced similar patterns after gel electrophoresis, with minor differences in molar ratios after being treated with the restriction enzymes suggesting that RS virus DNA obtained by superinfection of Raji cells is basically identical to HR-1 virus DNA but may contain a population of DNA a little more heterogenous than HR-1 virus DNA.     

Identification,mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus

The thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) was identified by biochemical transformation of 3T3 TK negative (TK⁻) to 3T3 TK positive (TK⁺) cells using specific viral DNA sequences. DNA fragments of the viral genome used in this study were obtained from a defined gene library of FLDV genome containing the complete viral DNA sequences. The selection of the converted cells was carried out under the condition of the HAT selection procedure. The results of these experiments revealed that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611 to 0.718 map units) is able to transform 3T3 TK⁻ to 3T3 TK⁺ cells. Additional experiments using the subclones of EcoRI DNA fragment C revealed that DNA sequences of 4.1 kbp size between the coordinates 0.669 to 0.718 of the FLDV genome possessed the ability for biochemical transformation, indicating that the TK gene locus is located in this particular region.     

Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32EcoRI CIV DNA fragments), was used for transfection ofChoristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring theEcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535–0.582 m.u.), M (7.25 kbp, 0.310–0.345 m.u.), O (6.5 kbp, 0.196–0.228 m.u.), Q (5.9 kbp, 0.603–0.631 m.u.), and Y (2.0 kbp, 0.381–0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of theEcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within theEcoRI DNA fragments C and H at the genome coordinates 0.924–0.930 and 0.535–0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of theEcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661–675 and 677–691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of theEcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535–0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within theEcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661–691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within theEcoRI CIV DNA fragment Y.     

Transformation of a rat cell line by an adenovirus type 12 DNA fragment

Attempts were successfully made to transform 3Y1 cells, a clonal cell line derived from a Fischer rat embryo, with an endonuclease R, EcoRI, fragment (EcoRI-C) of adenovirus (Ad) type 12 (16% of whole Ad12 DNA) as well as with whole Ad12 DNA. The transformed cell lines were positive for T antigen in immunofluorescence and for the viral DNA sequences by DNA-DNA reassociation kinetics.     

The oncogenicity of avian adenoviruses II. The arrangement of the viral DNA in tumors and transformed cells

The arrangement of the viral DNA in six independent hamster tumors (12–920 molecules per diploid equivalent of cellular DNA) and one rat transformed cell line (34 molecules per diploid equivalent of cellular DNA) induced by CELO virus was investigated. The DNAs from these tumors and the cell line were cleaved with restriction endonuclease EcoRI or HindIII, subjected to gel electrophoresis, and analyzed by Southern's blotting hybridization technique using radioactive EcoRI fragments of the viral DNA as the probe. These experiments indicated the following: (1) Most, if not all, of the viral DNA in each of these tumors and the cell line was integrated into cellular DNA. In two tumors (Group A), at least EcoRI-B (one of the terminal fragments of the viral DNA) and EcoRI-G (an internal fragment) were completely missing, whereas all of the EcoRI fragments were present in the others (Group B). (2) Most of the integrated viral DNA molecules of Group B were colinear with the DNA from the virus particle, and integration sites of the viral DNA to cellular DNA were confined within two segments of the viral DNA that comprised the terminal 4% of the total length at each end of the molecule. (3) There was relatively little variation of the site in the viral DNA sequence and of the site in the cellular DNA sequence at which integration took place; this was true even in tumor cells carrying hundreds of copies of the viral DNA. Such integrated viral DNA molecules with their flanking cellular DNA sequences formed “amplification units”, and which could be replicated many hundreds of times within a given cell. (4) The tumors and transformed cell line in Group B were, at least partially, different from each other with respect to the varieties of amplification units they carried.     

Herpesvirus-associated nuclear antigen(s) in cells biochemically transformed by fragments of herpesvirus DNA and in somatic cell hybrids

Human and mouse cells biochemically transformed by ultraviolet light (UV)-irradiated HSV-1 express HSV-1 thymidine kinase (TK) activity and also express type-specific herpesvirus-associated nuclear antigen(s) (HANA). To identify the HSV-1 DNA sequences coding for HANA and their location on the viral genome, studies were carried out on: (i) somatic cell hybrid clones obtained by fusing mouse [LM(TK^?)] cells with UV-irradiated HSV-1-transformed human [HeLa(BU25)/KOS 8-1] cells; and (ii) LM(TK^?) cells biochemically transformed with restriction endonuclease fragments of DNA which code for HSV-1 TK. Molecular hybridization experiments were also carried out and demonstrated that HSV-1 DNA sequences coding for TK were integrated in the biochemically transformed cells. The human-mouse somatic cell hybrid clones (LH81) which were HSV-1 TK+ were also HANA⁺, while clones counterselected in bromodeoxyuridine which had lost HSV-1 TK activity and DNA sequences likewise lost HANA. Previous studies had shown that the HSV-1 TK gene of LH81 hybrid clones was associated with a marker chromosome, designated M7, which consists of a human chromosome 17 translocated to the short arm of chromosome 3, or a modified M7 chromosome containing a translocation from a mouse chromosome. The present results indicate that at least one HANA gene was integrated in the same chromosome as the HSV-1 TK gene. LM(TK^?) cells biochemically transformed by HSV-1 DNA restriction nuclease fragments of diminishing size, which map in the HpaI-I region (26.2 to 31.7) of the HSV-1 genome, were HANA⁺ as well as TK⁺. The HANA⁺ cells included LM(TK^?)/TF pAGO PP and LM(TK^?)/TF pAGO PS clones. The latter are clones of LM(TK^?) cells biochemically transformed, respectively, by a PvuII fragment (1.35 × 10⁶ daltons) and a PvuII-SmaI fragment (0.9 × 10⁶ daltons; 30.2 to 31.1 map units) of HSV-1 DNA derived from Escherichia coli plasmid, pAGO. Since the PvuII-SmaI DNA fragment has only enough genetic information to code for a polypeptide of about 53,000 daltons and the HSV-1 TK polypeptide is about 40,000 daltons, the findings indicate that the genes for HSV-1 TK and one herpesvirus-associated nuclear antigen are either contiguous or overlapping, or HSV-1 TK and one HANA gene are identical.     

The structure of cauliflower mosaic virus. II. Identity and location of the viral polypeptides.

Adenovirus type 12 (Ad 12) tumor-specific transplantation antigen (TSTA) and the surface (S) antigen were examined using rat cells transformed with Ad 12 DNA and its fragments. WY3 (3Y1 cells transformed with Ad 12 whole DNA), CY1 (3Y1 cells transformed with the EcoRI-C fragment of Ad 12 DNA), and GY cells (3Y1 cells transformed with the HindIII-G fragment of Ad 12 DNA) contained TSTA and S antigen, but HY cells (3Y1 cells transformed with the BpaI-H fragment of Ad12 DNA) did not. These results suggest that TSTA and S antigens contain a protein(s) coded for by a portion of the transforming gene.     

Intracellular forms of adenovirus DNA. VII. Excision of viral sequences from cellular DNA in adenovirus type 2-infected KB cells

The high molecular weight DNA from adenovirus type 2-infected KB cells contains viral sequences covalently linked to cell DNA. These viral sequences have been analyzed after excision from the cellular DNA using three restriction endonucleases with different specificities, Eco RI, Bam HI, and Sal I. Upon cleavage of the high molecular weight DNA from adenovirus type 2-infected KB cells with these enzymes and subsequent analysis of the generated fragments by gel electrophoresis and DNA-DNA hybridization, viral DNA sequences are found which do not coincide in size with any of the fragments of adenovirus type 2 DNA generated by the same enzyme. These viral sequences are either larger than the largest restriction endonuclease fragment of adenovirus type 2 DNA, or they are intermediate in size to virion DNA fragments. Viral sequences are also found in size classes identical to those of the corresponding virion DNA fragments. These results indicate that adenovirus DNA sequences are present in the cellular DNA from adenovirus type 2-infected cells and that these viral sequences are covalently linked to cellular DNA. The pattern in which viral sequences are found to be distributed upon cleavage with restriction endonucleases and analysis by gel electrophoresis followed by DNA-DNA hybridization to specific fragments of adenovirus type 2 DNA suggests that adenovirus type 2 DNA is integrated in fragments rather than as the intact molecule.     

Integration and methylation of shope papilloma virus DNA in the transplantable V×2 and V×7 rabbit carcinomas

The Shope papilloma virus (SPV) DNA present in SPV-induced benign and malignant rabbit tumors, particularly in the transplantable carcinoma Vx2 and Vx7, was examined with regard to physical states and extent of methylation. Vx2 and Vx7 carcinomas contained 10–22 viral genomes per diploid cell, and domestic and cottontail rabbit papillomas 40–400 and 1000–8000; respectively. The digestion of Vx2 and Vx7 DNA with the restriction enzyme KpnI, which does not cleave SPV DNA, yielded a single virus-specific DNA band about nine times larger than the genome length, but EcoRI, which cuts the circular SPV DNA once, cleaved this DNA to the genome-size fragments. However, three or four weak bands which may contain viral segments linked to cellular sequences were also identified, and at least two were shared by both Vx2 and Vx7 carcinomas. The analysis with a set of MspI HpaII, which discriminates the methylated DNA sequence -CC¹GG-, showed that 10–40% of the sites of viral DNA are methylated in papillomas, 30–80% in primary carcinomas, and more than 90% in the transplantable carcinomas.     

Re-evaluation of a case of progressive multifocal leukoencephalopathy previously diagnosed as simian virus 40 (SV40) etiology

A case of progressive multifocal leukoencephalopathy (PML) reported previously to be of simian virus (SV40) etiology was re-evaluated. The supernatant from a 10% homogenate of brain material was inoculated into African green monkey kidney cells and BSC-1 cells which are permissive for SV40. However no cytopathic effect (CPE) developed and no virus was isolated. The brain supernatant agglutinated human group O erythrocytes and contained 5120 units/mL. The Hirt supernatant from the brain contained three DNA bands corresponding to forms I, II and III of circular double-stranded viral DNA. Restriction endonuclease cleavage analysis revealed that this viral DNA was different from SV40 DNA, but similar to JC virus DNA. After cloning of this viral DNA into pBR322 at the BamHI site, DNA homology of this virus and of SV40 was investigated. The cloned DNA hybridized with all four HpaI/EcoRI fragments of SV40 genome at the effective temperature (Tm) of ?50°C. At Tm ?28°C, however, the cloned DNA hybridized with only HpaI/EcoRI fragment B of the SV40 genome. In contrast to this, JC virus DNA hybridized with all five EcoRI/BamHI/HindIII fragments of cloned DNA even at Tm ?28°C. Therefore, the causative agent of this PML case was not SV40 but JC virus.     

Identification of adenovirus-type 12 gene products involved in transformation and oncogenesis

Primary baby rat kidney cells were transfected with Ad12 DNA fragments EcoRI C (left-terminal 16%) and HindIII G (left-terminal 7.2%), and the resulting transformed cells were established as cell lines. Injection of newborn hamsters with purified Ad12 DNA resulted in the induction of tumors in 2 out of 59 animals. A number of the in vitro transformed cells and a cell line derived from a tumor were found to contain DNA sequences homologous to the fragments used for transfection or tumor induction, and to express virus-specific RNA and T antigens. The cell lines were also studied with respect to their tumorigenicity in nude mice or hamsters. It was found that cells transformed by Ad12 EcoRI C, or by intact DNA or virus, were tumorigenic whereas cells transformed by fragment HindIII G were unable to induce tumors. To correlate this result with the presence or absence of viral gene products, virus-specific T antigens were identified by immunoprecipitation. From lytically infected cells major proteins of 60, 41, 19, 14.5, and 13.5 kD were precipitated. Cells transformed by fragment EcoRI C or intact viral DNA contained proteins of 60, 41, and 19 kD and of 50 and 36 kD. HindIII G-transformed cells contained proteins of 41 and 19 kD. Studies of the T antigens in two-dimensional gels and by tryptic peptide analysis have indicated that two virus-specific 60-kD proteins are expressed in Ad12-infected cells. The major protein probably represents the single-stranded DNA-binding protein encoded by region Ell (Ell-60 kD), while the minor protein represents a region EI-specific T antigen (EI-60 kD) encoded by region EIb. The 41-kD protein is encoded by region EIa and 19 kD by EIb. Our results suggest that expression of the EI-60-kD protein is required for oncogenicity in nude mice, but not for morphological transformation.