The mechanism of viral carcinogenesis by DNA mammalian viruses. VI. A new class of virus-specific RNA molecules in cells transformed by group C human adenoviruses

A new class of virus-specific RNA molecules was found in cells transformed by group C human adenovirus types 2, 5, and 6. RNA isolated from virus-free rat embryo cells transformed by adenovirus 2, 5, and 6 hybridized with all group C adenovirus DNA's (adenovirus 1, 2, 5, and 6) equally well, but not appreciably with group A and B adenovirus DNA's. Most likely no viral genes common to group A, B, and C adenoviruses are transcribed in adenovirus-transformed cells.Group C adenoviruses are closely related since they share 83 to 93 per cent of their base sequences as shown by DNA-DNA homology measurements. Group C DNA's share only 10 to 26 per cent of their base sequences with group A and B DNA's. Moreover, the shared sequences are not transcribed detectably in adenovirus transformed cells.Virus-specific RNA isolated from group C transformed cells contains 49 to 51 per cent G + C, but viral DNA's possess a 7 to 9 per cent higher G + C content. These differences suggest that only a portion of the viral genome with an average G + C content of 49 to 51 per cent is transcribed in group C adenovirus transformed cells.     

Synthesis of Complementary RNA Sequences During Productive Adenovirus Infection

Liquid RNA.DNA hybridization with separated strands of adenovirus type 2 DNA revealed that late nuclear RNA can hybridize to about 85% of the 1-strand and 10-15% of the h-strand, whereas late cytoplasmic RNA hybridizes to 65-70% and 25% of the l- and h-strand, respectively. With separated strands from the six EcoRI fragments of adenovirus type 2 DNA as probes, it was shown that late nuclear RNA hydridizes to 85-90% of the l-strand from all six EcoRI fragments. Since late cytoplasmic RNA hybridizes to 40-50% of the h-strand from both fragments EcoRI-B and EcoRI-C, complementary viral RNA sequences are synthesized during adenovirus infection. Complementarity between nuclear and cytoplasmic RNA could also be demonstrated by showing that late cytoplasmic RNA which had been preincubated with late nuclear RNA hybridized to a smaller fraction of the h-strand of fragment EcoRI-C than without preincubation. Double-stranded RNA which contains sequences that correspond to at least 60% of the viral genome was isolated from infected cells. However, less than 2% of the newly synthesized late RNA became double-stranded after incubation under annealing conditions, which suggests that RNA derived from one of the strands is present at a low concentration. Accordingly, it was shown that nearly all viral cytoplasmic RNA which is synthesized late after infection is derived from the l-strand.     

Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome.

The bulk of late virus-specific RNA synthesized in polyoma virus-infected mouse cells is larger than a single strand of poloma DNA. The arrangement of viral nucleotide sequences in these giant polyoma RNAs was studied by electron microscopy of hybrids between purified high molecular weight viral RNA and the HindII-1 fragment of polyoma DNA, which contains 91% of the viral genome. Hybrid molecules containing a short single-stranded gap (corresponding to the 9% of viral sequences not present in HindII-1), flanked by double-stranded regions, were photographed and measured. The majority of hybrid molecules contained no single-stranded loops or branches, showing that all viral sequences are transcribed contiguously and that no nonviral sequences are present in the RNA. Hybrid molecules, containing RNA up to 3.5 times the genome length, had a repeating structure of single-stranded gaps 8% of genome length interspersed with double-stranded regions 89% of genome length, showing that giant polyoma RNAs contain tandem repeats of the nucleotide sequence of the entire viral DNA. A small proportion of hybrid molecules contained single-stranded branches or deletion loops in characteristic positions, indicating that RNA "splicing" may occur on high molecular weight nuclear polyoma RNA.     

Biochemical Studies on Adenovirus Multiplication, XIX. Resolution of Late Viral RNA Species in the Nucleus and Cytoplasm

Late after infection of cultured human cells (KB) with adenovirus type 2, the nucleus contains heterogeneous viral RNA species ranging in size from 10 to 43 S. Four viral RNA species found in the nucleus (36, 38, 40, and 43 S) are synthesized predominantly during a 15-min labeling period with [(3)H]uridine, while smaller RNA species accumulate when labeling is continued for longer periods. In contrast, 6-8 viral RNA species, of sedimentation coefficient from 10 to 29 S, are found in the cytoplasm after a 30-min pulse label and a 2-hr chase. DNA-RNA hybridization-competition experiments demonstrate that viral RNA sequences present in nuclear 36-43S RNA are also present in cytoplasmic and polyribosomal RNA, suggesting that at least some of the cytoplasmic viral-specific RNA molecules are derived by cleavage of high molecular weight precursors from the nucleus.     

Endogenous Guinea Pig Virus: Equability of Virus-Specific DNA in Normal, Leukemic, and Virus-Producing Cells

The kinetics of hybrid formation between the RNA of BrdU-activated endogenous guinea pig virus and the DNA of leukemic, normal, or BrdU-activated guinea pig cells were measured by the technique of RNA.DNA hybridization in DNA excess. The results suggest that virus-specific sequences representing some 60-70% of the viral genome are unique (2-3 copies per haploid cell genome), while the remainder (30-40%) are reiterated (147 copies), and that the reiterated virus-specific DNA may be composed of more than one species, each having a different reiteration frequency. No difference was found in the quantity of viral DNA sequences contained in normal, leukemic, or bromodeoxyuridine-activated guinea pig cells. These data are considerably different from those reported for exogenous (infectious) oncornaviruses, where cells infected or transformed by exogenous RNA tumor viruses have been shown to contain increased amounts of virus-specific DNA. The data reported here are consistent with the contention that preexisting viral genes are activated by bromodeoxyuridine treatment. Results of hybridization experiments done at different DNA/RNA ratios suggest that although the virus-specific DNA is partly unique and partly reiterated, the viral RNA does not contain any detectable internal reiteration. Total mass of the viral RNA sequences is around 0.7 to 1 x 10(7) daltons.     

Viral DNA in Transformed Cells. III. The Amounts of Different Regions of the SV40 Genome Present in a Line of Transformed Mouse Cells

(32)P-Labeled SV40 DNA was treated sequentially with restricting endonucleases EcoRI and Hpa I, and the resulting four fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete SV40 DNA were measured in the presence of DNA extracted from the SVT2 line of SV40-transformed mouse cells. It was found that these cells contain about six copies of a segment of DNA which includes the early region of the SV40 genome, and about one copy of the late viral sequences.To map the region of the viral genome which is transcribed in SVT2 cells, separated strands of each of the four fragments were prepared and hybridized to total transformed cell RNA. Part of the E strands of the two DNA fragments (A and C) which span the early region of the SV40 genome were found to enter the hybrid.     

Immunologic selection against simian virus-40 transformed cells: Concomitant loss of viral antigens and early viral gene sequences

A clonal line of highly oncogenic "spontaneously transformed" mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10(4)-10(5)) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.     

Evidence That the AKR Murine-Leukemia-Virus Genome Is Complete in DNA of the High-Virus AKR Mouse and Incomplete in the DNA of the “Virus-Negative” NIH Mouse

The AKR mouse has a high titer of murine leukemia virus early in life, and virus-negative cells derived from embryos of this mouse strain can be activated to yield murine leukemia virus by treatment with 5-iododeoxyuridine. In contrast to this high-virus strain, the NIH Swiss mouse has a low incidence of leukemia and no murine leukemia virus has been isolated from it (virus-negative). We have investigated this difference between AKR and NIH mice by examining the sequences specific for murine leukemia virus in nucleic acids of these mice. A single-stranded viral-DNA probe synthesized in vitro using murine-leukemia-virus from the AKR mouse contains at least 87% of the sequences present in the 70S viral RNA; most of these sequences are in proportions similar to their content in the 70S RNA. Using this probe in nucleic acid hybridization experiments, we have shown that NIH-mouse-cell DNA and AKR-mouse-cell DNA differ with respect to sequences specific for AKR murine-leukemia-virus: NIH-mouse-cell DNA lacks some of the virus-specific sequences present in AKR-mouse-cell DNA, and there are two distinct sets of virus-specific sequences in AKR-mouse-cell DNA, whereas there is only one set in NIH-mouse-cell DNA.RNA from virus-negative AKR-mouse cells grown in tissue culture contains some, but not all, virus-specific RNA sequences; however, within 48 hr after initiating treatment of these cells with 5-iododeoxyuridine, the complete viral genome is represented in cellular RNA.     

The mechanism of viral carcinogenesis by DNA mammalian viruses: RNA transcripts containing viral and highly reiterated cellular base sequences in adenovirus-transformed cells (DNA-RNA hybridization-viral-cell mRNA)

Virus-specific RNA was isolated from cells transformed by human adenovirus 2 and 7 by multiple hybridizations with and elutions from homologous viral DNA; RNA molecules purified by this selection procedure hybridized efficiently with both viral DNA (24-50%) and DNA from untransformed cells (12-27%). Virus-specific RNA isolated in the same manner from cells productively infected with adenoviruses did not hybridize significantly with cellular DNA. These findings suggest that RNA molecules containing covalently-linked viral and cellular sequences are transcribed in cells transformed by human adenoviruses. The high efficiency of hybridization with DNA from untransformed cells implies that viral DNA is integrated adjacent to highly reiterated cellular DNA sequences.     

Quantitative and qualitative differences in DNA complementary to avian myeloblastosis virus between normal and leukemic chicken cells.

Hybridization of avian myeloblastosis virus (AMV) RNA with DNA immobilized on filters or in liquid with a vast DNA excess was used to measure the viral specific DNA sequences in chicken cells. Newly synthesized viral DNA (v-DNA) appears within an hour after infection of chicken embryo fibroblasts (CEF) with avian oncornaviruses. A fraction of newly synthesized v-DNA becomes integrated into the cellular genome and the remainder gradually disappears. A covalent linkage between v-DNA and cellular DNA was demonstrated to exist in CEF and in leukemic myeloblasts by alkaline sucrose velocity sedimentation. Hybridization of AMV RNA in DNA excess has revealed that there are 2 clases of viral specific sequences within normal as well as in leukemic cells. The 2 types of sequences differ in their rate of hybridization. The amount of both types of DNA sequences is about 2 times higher in leukemic cells than in normal cells. Both the fast- and slowly reacting sequences in leukemic cells exhibit a higher Tm (2 degrees C) than the respective DNA sequences in normal cells. Furthermore, when nucleotide sequences in AMV RNA complementary to normal DNA are removed first by exhaustive hybridization with normal DNA, the residual RNA only hybridizes with leukemic DNA but not with normal DNA. These results suggest that leukemic cells contain viral specific DNA sequences which are absent in normal cells. Endogenous v-DNA has been shown to be integrated in cellular DNA region(s) with a reiteration frequency of approximately 1,200 copies per cell and each integration unit appears to have a size approximately equivalent to the 35S RNA subunit of the viral genome. Viral sequences acquired after infection appear to be integrated in the unique region of cell DNA, or in tandem with the endogenous viral sequences.     

Effect of Poliovirus Double-Stranded RNA on Viral and Host-Cell Protein Synthesis

Cell-free protein-synthesizing systems that initiate on endogenous messenger RNA have been developed from uninfected and poliovirus-infected HeLa cells. Poliovirus double-stranded RNA is an effective inhibitor of protein synthesis in these extracts, and both cell-directed and virus-specific protein synthesis are equally sensitive to the inhibitory action of double-stranded RNA. The concentrations of double-stranded RNA required for inhibition are not achieved in the infected cell at early times after infection when host-cell shut-off occurs, but rather are achieved only late in infection when virus-specific protein synthesis begins to decline. This indicates that double-stranded RNA does not act as a direct agent to inhibit host cell protein synthesis following infection by poliovirus. The possible significance of inhibition by double-stranded RNA of poliovirus-specific protein synthesis is discussed.