Ribonucleotide sequence homology among avian oncornaviruses.

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between 3H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-3, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of 3H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as effeciently (100%) as by unlabeled AMV RNA. Hybridization between 3H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between 3H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between 3H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNA'S. Hybridization between 3H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 TO 26% BY RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybricization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Presence in Leukemic Cells of Avian Myeloblastosis Virus-Specific DNA Sequences Absent in Normal Chicken Cells

(3)H-labeled 35S RNA from purified avian myeloblastosis virus (AMV) was exhaustively hybridized with an excess of normal chicken DNA to remove all viral RNA sequences which are complementary to DNA from uninfected cells. The [(3)H]RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded [(3)H]RNA. The residual RNA hybridized to leukemic chicken DNA but did not rehybridize with normal chicken DNA. This demonstrates conclusively that DNA from AMV-induced leukemic cells contain viral-specific sequences which are absent in DNA from normal cells.     

Acquisition of viral DNA sequences in target organs of chickens infected with avian myeloblastosis virus.

The distribution of oncornavirus DNA sequences in various tissues of normal chickens and of chickens with leukemia or kidney tumors induced by avian myeloblastosis virus (AMV) was analyzed by DNA-RNA hybridization using 35S AMV RNA as a probe. All the tissues from normal chickens which were tested contained the same average cellular concentration of endogenous oncornavirus DNA. In contrast, different tissues from lekemic chickens and from chickens bearing kidney tumors contained different concentrations of AMV homologous DNA: in some tissues there was no increase whereas other tissues acquired additional AMV-specific DNA sequences. The increase was the greatest in tissues which can become neoplastic after infection, such as myeloblasts, erythrocytes, and kidney cells. It was directly demonstrated that DNA from AMV-induced kidney tumor contains AMV sequences which are absent in DNA from normal cells. A similar finding had been previously obtained with leukemic cells (15). 3H-labeled 35S RNA from purified AMV was exhaustively hybridized with an excess of normal chicken DNA to remove all the viral RNA sequences which are complementary to DNA from uninfected cells. The 3H-labeled RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded RNA. The residual RNA hybridized to chicken kidney tumor DNA but did not rehybridize with normal chicken DNA.     

Ribonucleotide Sequence Homology Among Avian Oncornaviruses

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between ³H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-0, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of ³H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as efficiently (100%) as by unlabeled AMV RNA. Hybridization between ³H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between ³H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between ³H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNAs. Hybridization between ³H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 to 26% by RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybridization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0 are different from the sequences shared by AMV and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Homology between avian oncornavirus RNAs and DNA from several avian species.

3H-labeled 35S RNA from avian myeloblastosis virus (AMV), Rous associated virus (RAV)-0, RAV-60, RAV-61, RAV-2, or B-77(w) was hybridized with an excess of cellular DNA from different avian species, i.e., normal or leukemic chickens, normal pheasants, turkeys, Japanese quails, or ducks. Approximately two to three copies of endogenous viral DNA were estimated to be present per diploid of normal chicken cell genome. In leukemic chicken myeloblasts induced by AMV, the number of viral sequences appeared to have doubled. The hybrids formed between viral RNA and DNA from leukemic chicken cells melted with a Tm 1 to 6 C higher than that of hybrids formed between viral RNA and normal chicken cell DNA. All of the viral RNAs tested, except RAV-61, hybridized the most with DNA from AMV-infected chicken cells, followed by DNA from normal chicken cells, and then pheasant DNA. RAV-61 RNA hybridized maximally (39%) with pheasant DNA, followed by DNA from leukemic (34%), and then normal (29%) chicken cells. All viral RNAs tested hybridized little with Japanese quail DNA (2 to 5%), turkey DNA (2 to 4%), or duck DNA (1%). DNA from normal chicken cells contained only 60 to 70% of the RAV-60 genetic information, and normal pheasant cells lacked some RAV-61 DNA sequences. RAV-60 and RAV-61 genomes were more homologous to the RAV-0 genome than to the genome of RAV-2, AMV, or B-77(s). RAV-60 and RAV-61 appear to be recombinants between endogenous and exogenous viruses.     

Quantitative Determination and Location of Newly Synthesized Virus-Specific Ribonucleic Acid in Chicken Cells Infected with Rous Sarcoma Virus

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs(2)SO(4) gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with (3)H-uridine could be quantitated by the addition of an internal standard consisting of (32)P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with (3)H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.     

Distribution of Deoxyribonucleic Acid Complementary to the Ribonucleic Acid of Avian Myeloblastosis Virus in Tissues of Normal and Tumor-Bearing Chickens

(3)H-labeled 70S ribonucleic acid (RNA) from purified avian myeloblastosis virus (AMV) was used as a probe in deoxyribonucleic acid (DNA)-RNA hybridization experiments to detect the presence of DNA complementary to the AMV genome in various tissues from noninfected normal chickens and from chickens infected with AMV. There was a remarkable constancy in the average cellular concentration of virus-specific DNA found in every tissue from the same uninfected chicken, and even in different chickens from the same strain. In contrast, different tissues from chickens bearing AMV-induced kidney tumors (embryonal nephromas) revealed an unequal distribution in the average virus-specific DNA content per cell. The increase was limited to tumor cells and to tissues that contain target cells for AMV, i.e., red blood cells, kidney cells, and possibly leukocytes. The red blood cells from AMV-infected chickens suffering from acute myeloblastic leukemia, although producing no virus, contained as many viral genome equivalents per cell as did leukemic myeloblasts known to produce large quantities of AMV. An increased viral DNA content was observed in the target cells of chickens that did not show any sign of tumor formation 6 months after infection with AMV. This study demonstrates that vertically transmitted viral DNA is uniformly and stably distributed among all tissues of the offspring, but that horizontal infection after hatching results in an increase in viral DNA content only in some dividing, target tissues that may or may not give rise to neoplasias.     

Integrated State of Oncornavirus DNA in Normal Chicken Cells and in Cells Transformed by Avian Myeloblastosis Virus

The covalent linkage of oncornavirus-specific DNA to chicken DNA was investigated in normal chicken embryo fibroblasts (CEF) and in virus-producing leukemic cells transformed by avian myeloblastosis virus (AMV). The virus-specific sequences present in cellular DNA fractionated by different methods were detected by DNA-RNA hybridization by using 70S AMV RNA as a probe. In CEF and in leukemic cells, the viral DNA appeared to be present only in the nucleus. After cesium chloride-ethidium bromide density equilibrium sedimentation, the viral DNA was present as linear, double-stranded molecules not separable from linear chicken DNA. After extraction by the Hirt procedure, the viral DNA precipitated with the high-molecular-weight DNA. After alkaline sucrose velocity sedimentation, the viral DNA cosedimented with the high-molecular-weight cellular DNA. The results indicate that in both types of cells studied, the oncornavirus-specific DNA sequences were linked by alkali stable bonds to nuclear cellular DNA of high molecular weight and did not appear to be present in free form of any size.     

Identification of the avian myeloblastosis virus genome. II. Restriction endonuclease analysis of DNA from lambda proviral recombinants and leukemic myeoblast clones.

Two lambda proviral DNA recombinants were characterized with a number of restriction endonucleases. One recombinant contained a complete presumptive avian myeloblastosis virus (AMV) provirus flanked by cellular sequences on either side, and the second recombinant contained 85% of a myeloblastosis-associated virus type 1 (MAV-1)-like provirus with cellular sequences adjacent to the 5' end of the provirus. Comparing the restriction maps for the proviral DNAs contained in each lambda hybrid showed that the putative AMV and MAV-1-like genomes shared identical enzyme sites for 3.6 megadaltons beginning at the 5' termini of the proviruses with respect to viral RNA. Two enzyme sites near the 3'-end of the MAV-1-like provirus were not present in the putative AMV genome. We also examined a number of leukemic myeloblast clones for proviral content and cell-provirus integration sites. The presumptive AMV provirus was present in all the leukemic myeloblast clones regardless of the endogenous proviral content of the target cells or the AMV pseudotype used for conversion. Multiple cellular sites were suitable for integration of the putative AMV genome and the helper genomes. The proviral genomes were all integrated colinearly with respect to linear viral DNA.     

Interspersion of sequences in avian myeloblastosis virus rna that rapidly hybridize with leukemic chicken cell DNA.

Liquid hybridization of progressively smaller fragments (35S, 27S, 15.5S, 12.5S, and 8S) of poly(A)-selected avian myeloblastosis virus RNA with excess DNA from leukemic chicken myeloblasts revealed that all sizes of RNA contained sequences complementary to both slowly and rapidly hybridizing cellular DNA sequences. Apparently, the RNA sequences which hybridize rapidly with excesses of cellular DNA are not restricted to any one region of the avian myeloblastosis virus 35S RNA. Instead, they appear to be randomly distributed over the entire 35S avian myeloblastosis virus RNA molecule with some positioned within 200 nucleotides of the poly(A) tract at the 3' end of the RNA.     

Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T_m (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T_m which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.     

Separation of DNA Sequences Complementary to the RNA of Avian Myeloblastosis Virus from Chicken DNA by Alkaline Cesium Chloride Density Sedimentation

Density gradient sedimentation in alkaline cesium chloride of DNA from normal chicken embryos or leukemic myeloblasts fragmented to a size of 13S revealed that the DNA sequences complementary to 70S avian myeloblastosis virus RNA sedimented in the high guanine plus cytosine region ahead of the main peak of cellular DNA. When the DNA was fragmented into pieces of 6.6S there was a broader distribution of the DNA sequences complementary to the viral RNA. This technique could be employed as a step towards the isolation of DNA copies of the entire viral RNA genome from the mass of host cellular DNA.     

Expression of Endogenous Retroviral Genes in Leukemic Guinea Pig Cells

The expression of guinea pig retrovirus (5-bromodeoxyuridine[BUdR]-induced GPV) was studied in guinea pig L(2)C leukemic lymphoblasts by use of molecular hybridization of viral complementary DNA (cDNA) to cellular RNA. It was found that L(2)C leukemic lymphoblasts, leukemic spleen, and BUdR-induced virus-producing cells contain virus-specific RNA: 0.05% (800 to 960 copies per cell), 0.02% (360 copies per cell), and 0.3% (5,120 copies per cell), respectively. Adult normal liver and spleen, on the other hand, contain less than 0.2 copy of viral RNA per cell. Both BUdR-induced cells and L(2)C leukemic lymphoblasts contained 14S, 22S, 35S, and 70S RNA species of total and cytoplasmic virus-specific RNA as determined by sucrose velocity gradient analysis and hybridization of sucrose gradient fractions to cDNA. Virus-specific mRNA was identified in both BUdR-induced cells and L(2)C leukemic lymphoblasts by the criterion that it cosedimented with purified polyribosomes in a sucrose gradient and that it changed to a lower sedimentation value if polyribosomes were disaggregated with EDTA prior to centrifugation. Virus-specific mRNA obtained from either the polyribosome region of purified polyribosomes or the released messenger region of EDTA-disaggregated purified polyribosomes consisted of 14S, 20S, and 35S species in both BUdR-induced cells and L(2)C leukemic lymphoblasts. Hybridization of cDNA to the RNA of L(2)C leukemic lymphoblasts and BUdR-induced cells was essentially complete. Additionally, leukemic lymphoblast RNA could displace 95% of the hybridization of BUdR-induced GPV 70S RNA to guinea pig DNA. The midpoints of thermal denaturation of hybrids formed between GPV cDNA and the RNA of either L(2)C leukemic lymphoblasts or the 70S RNA of BUdR-induced GPV were both 89 degrees C in 2x concentrated 0.15 M NaCl plus 0.015 M sodium citrate. These results show that BUdR-induced GPV genes are essentially completely expressed in L(2)C leukemic lymphoblasts and that virus-specific mRNA is present, although fewer copies of RNA are present in L(2)C leukemic lymphoblasts than in BUdR-induced cells.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

The genome and the intracellular RNAs of avian myeloblastosis virus

Avian myeloblastosis virus (AMV) is an acute leukemia virus which causes a myeloblastic leukemia in birds and transforms myeloid hematopoietic cells in vitro. We have analyzed RNA from AMV virions and from AMV-transformed producer and nonproducer cells by gel electrophoresis followed by transfer to chemically activated paper and hybridization to several complementary DNA (cDNA) probes. Using a cDNA probe specific for AMV, we identified two RNA species of 7.2 and 2.3 kb, which were present in all AMV-transformed cells and in all AMV virion preparations examined. The 7.2 kb species, which is presumably the genome of AMV, appears to contain the entire retroviral gag gene and at least part of the pol gene, but lacks much (or all) of the env gene. Thus AMV differs from other acute leukemia viruses described to date, since the latter have genomes of 5.5 to 5.6 kb, have only part of the gag gene and lack pol sequences. The smaller RNA does not contain gag-, pol- or env-specific nucleotide sequences but does carry nucleotide sequences from both the 5' and 3' termini of the genome, suggesting that it may be a subgenomic mRNA. Both the 7.2 and 2.3 kb species were associated with the 70S RNA complex in virions. These results suggest that AMV, unlike other acute leukemia viruses, does not express its transforming gene via a gag-related "fusion" protein but rather as a (so far unidentified) protein translated from a subgenomic mRNA.     

Nucleotide Composition of RNA hybridized to Homologous DNA from Cells transformed by Avian Tumour Viruses

PART of the evidence which indicates that RNA tumour viruses replicate through a DNA intermediate¹ was the detection of DNA which is complementary to the viral RNA in leukaemic cells transformed by avian myeloblastosis virus (AMV)² and in cells transformed in vitro by avian sarcoma viruses, Schmidt-Ruppin (SR-RSV) and B-77 (ref. 3). If this DNA serves as a template for the viral RNA, it must be a copy of the entire viral genome. One of the necessary requirements for this function is that the homologous DNA has the same nucleotide composition as the viral RNA. In this study, the average base composition of the RNA which had been hybridized to homologous DNA from transformed cells was compared with the base composition of the input viral RNA. Two experimental conditions had to be met: (1) the recovery of all the ribonucleotides which had been hybridized and (2) the absence of partially hybridized ribonucleotide sequences. The first requirement called for the deletion of the treatment of DNA-RNA hybrids with pancreatic ribonuclease fraction A and ribonuclease T₁ which had been used in our previous experiments because such a treatment can cause the non-random loss of hybridized nucleotides⁴. The second requirement called for a hybridization and washing procedure in which only specifically hybridized ribonucleotide sequences would remain bound to the filters. Both of these conditions were met by using fragmented viral RNA and a modified washing procedure which excluded the use of ribonuclease. The results show that the average nucleotide composition of the hybridized RNA is identical to that of the input viral RNA.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Myeloblastosis Virus

Avian myeloblastosis virus (AMV) 4S RNA was tested for amino acid acceptor activity for 18 of the 20 amino acids. A nonrandom distribution of viral tRNAs was found compared with tRNA from normal liver or from AMV-infected leukemic myeloblasts, confirming previous reports. Methionine and proline tRNAs were considerably enriched, whereas glutamic acid, glutamine, serine, tyrosine, and valine tRNAs were markedly depleted in AMV relative to homologous cellular tRNAs. The seven AMV tRNAs with the greatest amino acid acceptance capacities, which were in order methionine, proline, lysine, arginine, histidine, isoleucine, and threonine tRNAs, were compared with homologous tRNAs from leukemic myeloblasts and liver by reversed-phase 5 chromatography. Of the 25 isoaccepting chromatographic fractions identified, no tRNA species unique to AMV was detected. Only methionyl-tRNA showed a substantial quantitative variation in isoaccepting species compared with the host cell. Thus, viral selectivity for amino acid-specific tRNAs is not, generally, paralleled by selectivity for individual isoaccepting tRNA species. Qualitative differences in arginyl- and histidyl-tRNA isoaccepting species were discovered in virus and leukemic myeloblasts compared with liver. This indicates the existence of structural differences in these tRNA species which could be related to virus replication or expression.