Rate of divergence of cellular sequences homologous to segments of Moloney sarcoma virus.

The RNA genome of the Moloney isolate of murine sarcoma virus (M-MSV) consists of two parts--a sarcoma-specific region with no homology to known leukemia viral RNAs, and a shared region present also in Moloney murine leukemia virus RNA. Complementary DNA was isolated which was specific for each part of the M-MSV genome. The DNA of a number of mammalian species was examined for the presence of nucleotide sequences homologous with the two M-MSV regions. Both sets of viral sequences had homologous nucleotide sequences present in normal mouse cellular DNA. MSV-specific sequences found in mouse cellular DNA closely matched those nucleotide sequences found in M-MSV as seen by comparisons of thermal denaturation profiles. In all normal mouse cells tested, the cellular set of M-MSV-specific nucleotide sequences was present in DNA as one to a few copies per cell. The rate of base substitution of M-MSV nucleotide sequences was compared with the rate of evolution of both unique sequences and the hemoglobin gene of various species. Conservation of MSV-specific nucleotide sequences among species was similar to that of mouse globin gene(s) and greater than that of average unique cellular sequences. In contrast, cellular nucleotide sequences that are homologous to the M-MSV-murine leukemia virus "common" nucleotide region were present in multiple copies in mouse cells and were less well matched, as seen by reduced melting profiles of the hybrids. The cellular common nucleotide sequences diverged very rapidly during evolution, with a base substitution rate similar to that reported for some primate and avian endogenous virogenes. The observation that two sets of covalently linked viral sequences evolved at very different rates suggests that the origin of M-MSV may be different from endogenous helper viruses and that cellular sequences homologous to MSV-specific nucleotide sequences may be important to survival.     

Characterization of Gazdar murine sarcoma virus by nucleic acid hybridization and analysis of viral expression in cells.

Gazdar murine sarcoma virus (Gz-MSV) and Moloney murine sarcoma virus (M-MSV) are closely related. The complete M-MSV-specific nucleic acid sequences constituted a major portion of Gz-MSV-specific sequences. The MSV-specific sequences in both Gz-MSV and M-MSV genomes shared homology with hamster leukemia virus nucleic acid sequences. Both rat cells (S+L+) and hamster (S+L-) cells expressed two viral proteins of 68,000 and 70,000 daltons. These proteins were immunologically related to p60 purified from m1 virions of M-MSV.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Nature and distribution of feline sarcoma virus nucleotide sequences.

The genomes of three independent isolates of feline sarcoma virus (FeSV) were compared by molecular hybridization techniques. Using complementary DNAs prepared from two strains, SM- and ST-FeSV, common complementary DNA'S were selected by sequential hybridization to FeSV and feline leukemia virus RNAs. These DNAs were shown to be highly related among the three independent sarcoma virus isolates. FeSV-specific complementary DNAs were prepared by selection for hybridization by the homologous FeSV RNA and against hybridization by fline leukemia virus RNA. Sarcoma virus-specific sequences of SM-FeSV were shown to differ from those of either ST- or GA-FeSV strains, whereas ST-FeSV-specific DNA shared extensive sequence homology with GA-FeSV. By molecular hybridization, each set of FeSV-specific sequences was demonstrated to be present in normal cat cellular DNA in approximately one copy per haploid genome and was conserved throughout Felidae. In contrast, FeSV-common sequences were present in multiple DNA copies and were found only in Mediterranean cats. The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene.     

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.     

Virus-Like 30S RNA in Mouse Cells

Uninfected JLS-V9 mouse cells are known to express high levels of viral sequences that hybridize to complementary DNA made by the BrdU-induced virus of JLS-V9 cells. The genome in the BrdU-induced virus has been found to consist mainly of an RNA species that migrates as 30S RNA material during electrophoresis through agarose gels. This virus-like 30S RNA, designated VL30 RNA, apparently represents a new class of endogenous defective retroviruses that are not generally evident because of their defectiveness and lack of biological function. Fingerprint analysis and hybridization studies show that VL30 RNA does not have homology with the standard nondefective murine leukemia viruses. Upon superinfection with a nondefective murine leukemia virus, or upon induction of endogenous virus with BrdU, VL30 RNA is rescued into virions by phenotypic mixing. When VL30 RNA is rescued by BrdU induction, the VL30 RNA is mainly organized as a 50S complex, but when VL30 is rescued by superinfection, VL30 is also found in 70S RNA. Rescued VL30 RNA sequences can be reverse transcribed by the virion-associated DNA polymerase in an endogenous reaction. Many mouse cells express the sequences, whereas heterologous cells such as rat or rabbit cells do not contain them. By using hybridization of a complementary DNA probe to cellular RNA immobilized on paper, no subgenomic RNA related to the VL30 RNA could be found in cells expressing the VL30 sequences. From 20 to 50 copies of these sequences were found to be contained in the mouse genome. VL30 RNA is probably present in most stocks of leukemia and sarcoma viruses made in mouse cells.     

Viral RNA content of bovine leukemia virus-infected cells

A bovine leukemia virus (BLV)-producing cell line, fetal lamb kidney cells infected with BLV (FLK) contains one or a few copies of BLV proviral DNA in its genome. These cells contain 0.002% of viral RNA which sediments, in a sucrose gradient, at about 35S and between 18S and 28S.In cattle affected by enzootic bovine leukosis, tumor cells and circulating lymphocytes also contain one or a few copies of BLV proviral DNA integrated in their genome. However, in all cases tested (except one), no viral RNA was detected in these cells in conditions where one or two copies of viral genomic RNA per cell would have been easily detected.     

Molecular evidence for a type C retrovirus etiology of the lymphoproliferative disease of turkeys.

Recently, we isolated from the blood of lymphoproliferative disease (LPD)-affected turkeys a type C retrovirus distinct from the avian leukosis-sarcoma virus complex and the reticuloendotheliosis virus group. We present molecular evidence for the implication of this virus in the LPD of turkeys. Using complementary DNA of LPD viral RNA, we found that the LPD viral genome is specifically and efficiently transcribed (2,500 copies per cell) in LPD tumor cells. Moreover, the LPD tumor cells contained newly inserted LPD viral information (5 to 10 copies per haploid genome), which was not present before the infection. From the absence of LPD virus-specific sequences in the normal cell genome of turkeys, it was concluded that the LPD virus is not an endogenous virus of turkeys. DNA-DNA annealing experiments revealed that the degree of sequence homology between LPD viral complementary DNA and cellular DNA of turkeys was not higher than that between LPD viral complementary DNA and cellular DNA of other species, thus indicating that the virus does not originate from turkeys.     

Vertebrate DNAs contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus.

Avian myeloblastosis virus contains a continuous sequence of approximately 1,000 nucleotides which may represent a gene (amv) responsible for acute myeloblastic leukemia in chickens. This sequence appears to have been acquired from chicken DNA and to be substituted for the envelope gene in the viral genome. We used hybridization probes enriched for the amv sequences and conditions that facilitate annealing of partially homologous nucleotide sequences to show that cellular sequences related to amv are present in the genomes of all vertebrates ranging from amphibians to humans but were not detected in fish, sea urchins, or Escherichia coli. In contrast to the preceding findings, nontransforming endogenous proviral nucleotide sequences closely related to the remainder of the avian myeloblastosis virus genome and to the entire myeloblastosis-associated helper virus are present only in chicken DNA. The amv-related cellular sequences appear to be highly conserved during evolution and to be contained at only one or a few locations in the genome of vertebrates. Within closely related species, they appear to share common evolutionary genetic loci. These findings and similar ones obtained with other highly oncogenic retroviruses containing a transforming gene suggest a general mechanism for acquisition of viral oncogenic sequences and an essential role for these sequences in the normal cellular state.     

Characterization of the oncogene (erb) of avian erythroblastosis virus and its cellular progenitor.

Avian erythroblastosis virus (AEV) induces primarily erythroblastosis when injected intravenously into susceptible chickens. In vitro, the hematopoietic target cells for transformation are the erythroblasts. Occasional sarcomas are also induced by intramuscular injection, and chicken or quail fibroblasts can be transformed in vitro. The transforming capacity of AEV was shown to be associated with the presence of a unique nucleotide sequence denoted erb in its genomic RNA. Using a simplified procedure, we prepared radioactive complementary DNA (cDNAaev) representative of the erb sequence at a high yield. Using a cDNAaev excess liquid hybridization technique adapted to defective retroviruses, we determined the complexity of the erb sequence to be 3,700 +/- 370 nucleotides. AEV-transformed erythroblasts, as well as fibroblasts, contained two polyadenylated viral mRNA species of 30 and 23S in similar high abundance (50 to 500 copies per cell). Both species were efficiently packaged into the virions. AEV-transformed erythroblasts contained additional high-molecular-weight mRNA species hybridizing with cDNAaev and cDNA5' but not with cDNA made to the helper leukosis virus used (cDNArep). The nature and the role, if any, of these bands remain unclear. The erb sequence had its counterpart in normal cellular DNA of all higher vertebrate species tested, including humans and fish (1 to 2 copies per haploid genome in the nonrepetitive fraction of the DNA). These cellular sequences (c-erb) were transcribed at low levels (1 to 2 RNA copies per cell) in chicken and quail fibroblasts, in which the two alleged domains of AEV-specific sequences corresponding to the 75,000- and 40,000-molecular-weight proteins seemed to be conserved phylogenetically and transcribed at similar low rates.     

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.     

Selective degradation of integrated murine leukemia proviral DNA by deoxyribonucleases

The sensitivity to micrococcal nuclease and DNAase I of the integrated proviral DNA sequences in Swiss mouse cells infected with Moloney murine leukemia virus has been studied. Chromatin was separated into micrococcal nuclease-sensitive and -resistant regions, and the amount of proviral sequences in these DNA preparations was estimated by kinetic hybridization with single-stranded complementary DNA of Moloney murine leukemia virus. At least two thirds of the proviral DNA sequences were found in the open regions of chromatin, and only one third was resistant to nuclease. The proviral DNA sequences are even more sensitive to deoxyribonuclease I. When intact nuclei were treated with limited amounts of enzyme, only 5% of the nuclear DNA was digested, whereas 48% of the proviral DNA was degraded.The proviral DNA sequences in cells which do not produce virus are more resistant to nuclease digestion, as compared to virus producer cells. Thus the endogenous proviral sequences, in normal uninduced Swiss mouse cells, are randomly distributed between resistant and sensitive portions of chromatin when tested with either micrococcal nuclease or pancreatic deoxyribonuclease I. The effect of cell cycle synchronization on the accessibility of the proviral sequences to pancreatic deoxyribonuclease I was investigated with rat cells infected with Moloney murine leukemia virus. The amount of proviral DNA sensitive to pancreatic deoxyribonuclease I is higher in actively dividing cells than in cells arrested at Go phase, which produce only small amounts of virus.     

Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.     

Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells

The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed.     

Mason-Pfizer virus RNA genome: relationship to the RNA of morphologically similar isolates and other oncornaviruses.

The 60-70S RNA of Mason-Pfizer virus (MPV) was iodinated in vitro and used in both direct and competitive molecular hybridization studies. MPV proviral sequences are present at a frequency of approximately one to two copies per haploid genome in the DNA of experimentally infected human cells. By nucleic acid competition hybridization, MPV RNA was found to be indistinguishable from the RNA of a virus (X381) isolated from a rhesus mammary gland and from RNA isolated from the cytoplasm of AO cells (Parks et al., 1973) and HeLa cells (Gelderblom et al., 1974), both previously reported to produce MPV-related particles. No homology was observed, however, between MPV RNA and the RNA, or the DNA, from two clones of HeLa cells obtained from the American Type Culture Collection. Hybridization of MPV 60-70S RNA to the DNA of normal tissues of humans and to the DNA of 11 other species revealed that MPV is not an endogenous virus of any of these species. Competition hybridization revealed no detectable sequence homology between the RNA of MPV and the RNAs of simian sarcoma virus, murine mammary tumor virus, murine leukemia virus, BUdR-induced guinea pig virus, or avian myeloblastosis virus. These nucleic acid studies substantiate previous ultrastructural and immunological findings that MPV and morphologically similar isolates constitute a distinct group of oncornavirus.