Mechanism of Viral Carcinogenesis by DNA Mammalian Viruses, VII. Viral Genes Transcribed in Adenovirus Type 2 Infected and Transformed Cells

DNA-RNA hybridization-competition experiments were used to compare the virus-specific RNA sequences synthesized during productive infection with human adenovirus type 2 with those synthesized in virus-free adenovirus type 2 transformed cells. The "early" virus-specific RNA present at six hours after infection, prior to the onset of viral DNA synthesis, represents 8-20 percent (2 to 10 genes) of the viral genome. All viral RNA sequences synthesized early are also present "late," at 18 hours after infection. The base sequences transcribed in transformed cells are homologous to approximately 50 per cent of the sequences transcribed early after infection. Thus only 4 to 10 per cent of the viral genome, representing 1 to 5 viral genes, are transcribed in adenovirus type 2 transformed cells. The virus-specific RNA synthesized 18 hours after infection was not found in transformed cells, suggesting that either these late viral genes are not present or are not transcribed in adenovirus type 2 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.     

Virus-Specific Messenger RNA and Nascent Polypeptides in Polyribosomes of Cells Replicating Murine Sarcoma-Leukemia Viruses

We present evidence that virus-specific RNA is present in polyribosomes of transformed cells replicating the murine sarcoma-leukemia virus complex and that it serves as messenger RNA for the synthesis of viral-coded proteins. Both virus-specific RNA (detected by hybridization with the [(3)H]DNA product of the viral RNA-directed DNA polymerase) and nascent viral polypeptides (measured by precipitation with antiserum to purified virus) were found in membrane-bound and free polyribosomes. Membrane-bound polyribosomes contained a higher content of both virus-specific RNA and nascent viral polypeptides. From 60 to 70% of viral RNA sequences were released from polyribosomes with EDTA, consistent with a function as messenger RNA. Maximum amounts of both virus-specific RNA and nascent viral polypeptides were found in the polyribosome region sedimenting at about 350 S.     

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.     

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.     

Isolation of RNA with Properties of Messenger RNA from Cerebral Polyribosomes

RNA which dissociated from purified cerebral polyribosomes of adult rats in the presence of EDTA was isolated by fractionation in a discontinuous sucrose gradient. The yield was 2% of the total polyribosomal RNA. The base composition resembled the complementary values for rat DNA and was very different from base compositions of ribosomal RNA and transfer RNA. This RNA fraction contained a large proportion of molecules which were rapidly labeled in vivo and hybridized to homologous DNA. The polyribosomal RNA preparation also exhibited high template activity in a cerebral cell-free system which had previously been stripped of the capacity to incorporate amino acids in the absence of added messenger RNA (mRNA). Sedimentation analysis revealed only two peaks, with coefficients of approximately 8 S and 16 S. The data indicate that RNA with the properties of mRNA can be selectively isolated from cerebral polyribosomes under mild conditions which avoid degradation.     

SV40-Specific RNA in the Nucleus and Polyribosomes of Transformed Cells

Cells transformed by the oncogenic virus SV40 are known to contain viral DNA integrated into cellular DNA and to produce virus-specific RNA. It has been shown that nuclear molecules containing virus-specific sequences are considerably longer than presumed virus-specific mRNA molecules from cytoplasmic polyribosomes. This finding suggests the possibility that cytoplasmic mRNA is derived by the specific cleavage of larger nuclear RNA.     

AKR Murine Leukemia Virus Genome: Frequency of Sequences in DNA of High-, Low-, and Non-Virus-Yielding Mouse Strains

Studies with a single-stranded DNA probe complementary to the RNA of mouse-tropic AKR murine leukemia virus indicate that the complete genome of the AKR-type murine leukemia virus is present in the DNA of high- and low-virus-yielding mouse strains, while DNA of non-virus-yielding strains contains only a part of the genome. Furthermore, in those strains where the genome is complete, two populations of virus-specific DNA sequences can be identified (more abundant and less abundant species) according to their rate of association with the probe. Low-virus-yielding mouse strains contain fewer copies of the less abundant species and, consequently, fewer complete viral genomes than do high-virus-yielding strains. Thus, in the ten strains tested, there is a good correlation between completeness of the genome of AKR-type murine leukemia virus in cellular DNA and the capacity of the cells to release infectious AKR-type murine leukemia virus. Moreover, the number of complete viral genomes correlates with the frequency of infectious virus production by virus-positive strains. DNA from wild Mus musculus also contained viral sequences, the sample tested showing reassociation kinetics identical to the non-virus-producing strains.     

Virus-Specific Messenger RNA on Free and Membrane-Bound Polyribosomes from Cells Infected with Rauscher Leukemia Virus

Cells infected by Rauscher leukemia virus synthesize virus-specific RNA which can be detected by hybridization to the single-stranded DNA copy of the viral RNA. Evidence is provided that virus-specific RNA is present in free and membrane-bound polyribosomes of these cells. The relative content of virus-specific RNA, as measured by hybridization, is 6-10 times less on free polyribosomes than on membrane-bound polyribosomes. The messenger RNA associated with both classes of polyribosomes was characterized by density gradient centrifugation. In addition to a major RNA species identified as 36S RNA, at least 2 minor components in the 14S and 21S region have also been found. There is a striking difference in the distribution of these RNA species between free and membrane-bound polyribosomes.     

Patterns of Simian Virus 40 DNA transcription after acute infection of permissive and nonpermissive cells

Small amounts of fractionated, denatured, (32)P-labeled DNA from SV40 virus were incubated with a large excess of the complementary RNA of SV40 prepared in vitro with Escherichia coli RNA polymerase; the viral DNA strands were separated on hydroxyapatite columns. The RNA present in green monkey cells late in the lytic cycle reacted with 40-42% of the strand complementary to the in vitro complementary RNA (minus strand), and 60-64% of the opposite (plus) strand. "Early lytic" RNA failed to significantly interact with the plus strand, but formed stable duplex molecules with 35-39% of the minus strand. The RNA prepared from mouse embryo cells 24 hr after infection with SV40 combined with 35-38% of the minus strand and 60-62% of the plus strand. In all cases, the same regions of either the plus or minus strand appear to be transcribed in permissive and nonpermissive infections.     

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.     

Polyadenylylated RNA complementary to a mouse retrovirus-like multigene family is rapidly and specifically induced by epidermal growth factor stimulation of quiescent cells.

Complementary DNA probes prepared from total polysomal poly(A)+RNA populations were used to identify clones of mouse DNA containing sequences whose expression is specifically enhanced after epidermal growth factor (EGF) stimulation of quiescent mouse embryo cells in culture. Three such clones were isolated and used to study changes in the levels of clone-specific poly(A)+RNA in the polysomes of cells after mitogenic stimulation by EGF. RNA complementary to sequences present in these clones increased approximately equal to 10-fold as a fraction of the total poly(A)+RNA by 6 hr after stimulation. All three clones were found by hybridization criteria to contain sequences related to the class of mouse retrovirus or transposon-like elements termed VL30. These VL30-related sequences were further found to be complementary to EGF-inducible poly(A)+RNAs and enhanced expression was detectable as early as 1 hr after EGF stimulation. In contrast, nine additional clones, including an AKR-type murine leukemia provirus DNA clone, contained no detectable VL30 sequence elements and were complementary to poly(A)+RNA species whose relative concentration was essentially constant in quiescent and EGF-stimulated cells. Therefore, VL30 sequence elements appear distinct in that they encompass members whose expression is specifically regulated in response to a defined peptide growth factor.