Nucleotide Sequence Relationships of Avian RNA Tumor Viruses: Measurement of the Deletion in a Transformation-Defective Mutant of Rous Sarcoma Virus

Stocks of cloned helper-independent Rous sarcoma virus (RSV) spontaneously segregate transformation-defective (td) mutants that appear to have an RNA genome composed of smaller subunits than those of the patent virus. Differential hybridization and competitive hybridization techniques involving reactions between viral RNA and proviral sequences in host cell DNA (under conditions of initial DNA excess) were used to measure the extent of the deletion in a td mutant of Prague strain (Pr) of RSV (Pr RSV-C). Viral 60 to 70S RNA sequences labeled to 1 to 5 x 10(7) counts per min per mug with (125)I were characterized with respect to their properties in hybridization reactions and used to reinforce data obtained with [(3)H]RNA of lower specific activity. By these techniques, about 13% +/- 3% of the sequences Pr RSV-C that formed hybrids with DNA from virus-induced sarcomas appeared to be deleted from the genome of td Pr RSV-C. Studies comparing hybridization of RNA from Pr RSV-C and td Pr RSV-C with RSV-related sequences in normal cells, and competition experiments with RNA from the endogenous chicken oncornavirus Rous-associated virus type 0 (RAV-0) provided evidence that the majority, if not all, of the RNA sequences of Pr RSV-C deleted from its transformation-defective mutant are not represented in normal chicken DNA. Competition studies with a leukosis virus, RAV-7, indicated this virus also lacks a genome segment of about the same size as the deletion in the td mutant. Finally, the genome of all three "exogenous" viruses was found to lack a small segment (about 12%) of sequences present in the endogenous provirus of RAV-O.     

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.     

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.     

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.     

Nucleotide Sequences Derived from Pheasant DNA in the Genome of Recombinant Avian Leukosis Viruses with Subgroup F Specificity

Recombination between viral and cellular genes can give rise to new strains of retroviruses. For example, Rous-associated virus 61 (RAV-61) is a recombinant between the Bryan high-titer strain of Rous sarcoma virus (RSV) and normal pheasant DNA. Nucleic acid hybridization techniques were used to study the genome of RAV-61 and another RAV with subgroup F specificity (RAV-F) obtained by passage of RSV-RAV-0 in cells from a ring-necked pheasant embryo. The nucleotide sequences acquired by these two independent isolates of RAV-F that were not shared with the parental virus comprised 20 to 25% of the RAV-F genomes and were indistinguishable by nucleic acid hybridization. (In addition, RAV-F genomes had another set of nucleotide sequences that were homologous to some pheasant nucleotide sequences and also were present in the parental viruses.) A specific complementary DNA, containing only nucleotide sequences complementary to those acquired by RAV-61 through recombination, was prepared. These nucleotide sequences were pheasant derived and were not present in the genomes of reticuloendotheliosis viruses, pheasant viruses, and avian leukosis-sarcoma viruses of subgroups A, B, C, D, and E. They were partially endogenous, however, to avian DNA other than pheasant. The fraction of these nucleotide sequences present in other avian DNAs generally paralleled the genetic relatedness of these avian species to pheasants. However, there was a high degree of homology between these pheasant nucleotide sequences and related nucleotide sequences in the DNA of normal chickens as indicated by the identical melting profiles of the respective hybrids.     

Influence of env and long terminal repeat sequences on the tissue tropism of avian leukosis viruses.

Adsorption and penetration of retroviruses into eucaryotic cells is mediated by retroviral envelope glycoproteins interacting with host receptors. Recombinant avian leukosis viruses (ALVs) differing only in envelope determinants that interact with host receptors for subgroup A or E ALVs have been found to have unexpectedly distinctive patterns of tissue-specific replication. Recombinants of both subgroups were highly expressed in bursal lymphocytes as well as in cultured chicken embryo fibroblasts. In contrast, the subgroup A but not subgroup E host range allowed high levels of expression in skeletal muscle, while subgroup E but not subgroup A envelope glycoproteins permitted efficient replication in the thymus. A subgroup B virus (RAV-2), like the subgroup E viruses, demonstrated a distinct bursal and thymic tropism, further supporting the theory that genes encoding receptors for subgroup B and E viruses are allelic. The source of long terminal repeats (LTRs) or adjacent sequences also influenced tissue-specific replication, with the LTRs from endogenous virus RAV-0 supporting efficient replication in the bursa and thymus but not in skeletal muscle. These results indicate that ALV env and LTR regions are responsible for unexpectedly distinctive tissue tropisms.     

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.     

Sequence of the Long Terminal Repeat and Adjacent Segments of the Endogenous Avian Virus Rous-Associated Virus 0

Rous-associated virus 0 (RAV-0), an endogenous chicken virus, does not cause disease when inoculated into susceptible domestic chickens. An infectious unintegrated circular RAV-0 DNA was molecularly cloned, and the sequence of the long terminal repeat (LTR) and adjacent segments was determined. The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1. Like the EV-1 LTR, the RAV-0 LTR is smaller (278 base pairs instead of 330) than the LTRs of the oncogenic members of the avian sarcoma virus-avian leukosis virus group. There is, however, significant homology. The most striking differences are in the U(3) region of the LTR, and in this region there are a series of small segments present in the oncogenic viruses which are absent in RAV-0. These differences in the U(3) region of the LTR could account for the differences in the oncogenic potential of RAV-0 and the avian leukosis viruses. I also compared the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences. A segment of approximately 200 bases to the right of the LTR (toward gag) is almost identical in RAV-0 and the Prague C strain of Rous sarcoma virus. The segment of RAV-0 which lies between the end of the env gene and U(3) is approximately 190 bases in length. Essentially this entire segment is present between env and src in the Schmidt-Ruppin A strain of Rous sarcoma virus. Most of this segment is also present between env and src in Prague C; however, in Prague C there is an apparent deletion of 40 bases in the region adjacent to env. In Schmidt-Ruppin A, but not in Prague C, about half of this segment is also present between src and the LTR. This arrangement has implications for the mechanism by which src was acquired. The region which encoded the gp37 portion of env appears to be very similar in RAV-0 and the Rous sarcoma viruses. However, differences at the very end of env imply that the carboxy termini of RAV-0, Schmidt-Ruppin A, and Prague C gp37s are significantly different. The implications of these observations are considered.     

Sequences outside of the long terminal repeat determine the lymphomogenic potential of Rous-associated virus type 1.

Recombinant avian leukosis viruses have been constructed from the molecularly cloned DNAs of Rous-associated virus type 1 (RAV-1) and Rous-associated virus type 0(RAV-0). Virus encoded by the cloned RAV-1 DNA induced a high incidence of B-cell lymphoma and a moderate incidence of a variety of other neoplasms. Virus encoded by the cloned RAV-0 DNA did not cause disease. Virus recovered from DNA constructions that encoded the gag, pol, and 5' env sequences of RAV-0 and the 3' env and long terminal repeat sequences of RAV-1 did not cause a high incidence of lymphoma. Rather, these constructed viruses induced a low incidence of a variety of neoplasms. Virus recovered from reconstructed pRAV-1 DNA had the same disease potential as did virus recovered from the parental pRAV-1 DNA. These results indicate that the long terminal repeat sequences of RAV-1 do not confer the potential to induce a high incidence of B-cell lymphoma.     

Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken.

Using less stringent hybridization conditions and cloned viral DNA probes representing the avian sarcoma virus gag, pol, env, and long terminal repeat (LTR) gene sequences, we detected related sequences in two avian species purportedly lacking all endogenous avian leukosis viruses, the ev- chicken and the Japanese quail. The blot hybridization patterns obtained with the various probes suggest the presence of between 40 and 100 copies of retrovirus-related sequences in the genomes of these two species. An ev- chicken genomic DNA library was prepared and screened with gag-specific and pol-specific DNA probes. Several different clones were obtained from this library and characterized. Analysis of these clones revealed that the retrovirus-related gene sequences are linked in the order LTR-gag-pol-env-LTR, a structure indicative of a complete provirus. These data indicate the presence of previously unidentified endogenous retrovirus species in avian cells, suggesting that under the appropriate conditions of hybridization additional, more distantly evolved families of endogenous retrovirus genes may be identified in vertebrate species.     

Purification of DNA complementary to nucleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses.

We have prepared radioactive DNA (cDNA_sarc) complementary to nucleotide sequences which represent at least a portion of the viral gene(s) required for neoplastic transformation of fibroblasts by an avian sarcoma virus. The genetic complexity of cDNA_sarc (~1600 nucleotides) is sufficient to represent an entire cistron. The genomes of three independent isolates of avian sarcoma viruses share nucleotide sequences closely related to cDNA_sarc, whereas the sequences are absent from transformation-defective mutants of avian sarcoma viruses, several avian leukosis viruses, a non-pathogenic endogenous virus of chickens (Rous-associated virus-O), sarcoma-leukosis viruses of mice and cats, and mouse mammary tumor virus. We conclude that the transforming gene(s) of all avian sarcoma viruses have closely related or common genetic lineages distinct from the transforming genes in sarcoma viruses of other species. Our results conform to previous reports that transformation-defective variants of avian sarcoma viruses are mutants with identical regions deleted from each subunit of a polyploid genome.     

Selective shedding and congenital transmission of endogenous avian leukosis viruses.

Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group-specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences.     

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.     

Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection.

We inoculated susceptible chicken embryos with the endogenous avian leukosis virus Rous-associated virus-0 (RAV-0) on day 6 of incubation. At 1 week after hatching, RAV-0-infected and control chickens were inoculated with either RAV-1 or RAV-2, exogenous viruses belonging to subgroups A and B, respectively. The chickens injected with RAV-0 as embryos remained viremic with exogenous virus longer and either failed to develop type-specific humoral immunity to exogenous virus or developed it later than the control chickens not inoculated with RAV-0. The RAV-0-injected chickens also developed neoplasms at a much higher frequency than did the control chickens. We suggest that the lower immune responses of the RAV-0-injected chickens were due to an immunological tolerance to envelope group-specific glycoproteins shared among endogenous and exogenous viruses.     

Lack of Sequence Homology Among RNAs of Avian Leukosis-Sarcoma Viruses, Reticuloendotheliosis Viruses, and Chicken Endogenous RNA-Directed DNA Polymerase Activity

The relatedness of the RNAs of the three avian systems, including six avian leukosis-sarcoma viruses, four reticuloendotheliosis viruses, and the microsome fraction of normal uninfected chicken embryo cells, containing RNA and a DNA polymerase have been studied by nucleic acid hybridization. All six avian leukosis-sarcoma viruses have closely related nucleotide sequences; and all four reticuloendotheliosis viruses have closely related nucleotide sequences. But, almost no similarities were detected between the RNAs of avian leukosis-sarcoma viruses and reticuloendotheliosis viruses. The RNA template of the endogenous RNA-directed DNA polymerase activity of normal uninfected chicken cells had no detectable relationship to RNAs of avian leukosis-sarcoma and reticuloendotheliosis viruses.     

Structural protein markers in the avian oncoviruses.

The proteins of purified avian oncoviruses were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing. Certain members of the avian leukosis-sarcoma viruses (ALSV) had group-specific antigens with altered electrophoretic properties. (i) The p27 protein of Rous-associated virus 0 (RAV-0) had a lower electrophoretic mobility in SDS gels and a lower isoelectric point than the p27 of other ALSV. (ii) The p19 proteins of RAV-1, RAV-2, and the Bryan high-titer strain of Rous sarcoma virus had higher mobilities in SDS gels than did the corresponding protein of other viruses. This altered electrophoretic mobility was correlated with specific differences in the tryptic peptides of radioiodinated p19s. (iii) The p15 protein of RAV-7 had a lower mobility in SDS gels than did the p15 of other ALSV. These markers were used in a study of the structural proteins of subgroup E RAV-60 produced after infection of chicken embryo cells by exogenous ALSV. Although exogenous group-specific protein markers could often be identified in the subgroup E isolates, one RAV-60 had a p27 that comigrated with the p27 of RAV-0. The p19s of two other RAV-60 isolates had electrophoretic properties that were different than those of p19s from either RAV-0 or the exogenous viruses. These results support the hypothesis that RAV-60 is generated by recombination between endogenous and exogenous oncoviruses and indicate that at least the p27 encoded by RAV-0 is closely related to a protein specified by endogenous viral information in chicken cells.     

Organization of shared and unshared sequences in the genomes of chicken endogenous and sarcoma viruses.

The genome of the genetically transmitted endogenous C type virus of chickens, RAV-O, is closely related to that of Rous sarcoma virus (RSV). Nevertheless, these viruses differ widely in oncogenicity and regulation by the host cell. Competitive hybridization analysis of ¹²⁵I-labeled genomic RNA demonstrated that the genome of RAV-O lacks about 35% of the sequences of nondefective RSV which formed hybrids with proviral DNA from RSV-infected cells, and that the genome of transformation-defective deletion mutants of RSV (td RSV) lacks about 15% of these sequences. Conversely, about 12% of the RAV-O sequences forming hybrids with normal chicken cell DNA were not detected in the sarcoma virus. A technique was developed to map the location of these unshared sequences by competitive hybridization. The deletion in the genome of td RSV was seen to begin at about 0.2 and to end at about 0.05 of the genome length from the 3′ end of sarcoma virus RNA, confirming the results of other laboratories using the method of mapping RNAase TI resistance of oligonucleotides. The 35% of RSV sequences missing and/or diverged in the genome of RAV-O were concentrated within 40% of the sarcoma virus genome from the 3′ end, and most of this large section did not appear to form hybrids with chicken DNA under the conditions of these experiments. A low level of hybrid formation was, however, detected between uninfected chicken cellular DNA and a small fraction of the nucleotides in the region of the td deletion. Analysis of RAV-O 3′ end fragments demonstrated that the genomic sequences of RAV-O missing in RSV were concentrated at the 3′ end of the endogenous viral genome. We conclude that the sequence differences between endogenous and sarcoma viruses are largely concentrated in specific regions of the viral genome.     

Viral DNA in bursal lymphomas induced by avian leukosis viruses.

Avian leukosis viruses (ALV) induce malignant lymphoma of the bursa of Fabricius. Viral DNA in tumors and normal tissues from infected birds were analyzed by using restriction endonucleases. Viral DNA fragments diagnostic of the exogenous ALV were easily detected in tumors, uninvolved bursal tissue, kidney, and erythrocyte nuclei. Exogenous viral DNA was more difficult to detect in liver. Using a restriction endonuclease (SacI) which cleaves linear unintegrated ALV DNA in a single site to define integration sites in DNA from the various tissues, we were able to detect ALV DNA only in tumor tissue. We concluded that the proviral DNA detected in the various nontumor tissue must be integrated in multiple sites. The appearance of ALV integration sites uniquely in tumors suggests that they are clonal growths. Furthermore, the data suggested the presence of a single exogenous integration site for the ALV provirus in each of six early neoplastic bursal nodules. This provirus appeared to retain the organization of EcoRI and BamHI recognition sequences present in the genome of virus used to infect the birds. The ALV integration site appeared different in each of the tumors studied. In a widespread metastatic lymphoma, multiple ALV integration sites were found as well as structural alterations in at least some copies of the ALV provirus.     

Lack of infectivity of the endogenous avian leukosis virus-related genes in the DNA of uninfected chicken cells.

The infectivity of the avian leukosis virus-related genes in the DNA of four genetically distinct types of chicken cells was determined. Infectious DNA of Rous-associated virus-O(RAV-O) was obtained from V- chicken cells which were experimentally infected with RAV-O and from V+tvbs chicken cells, which spontaneously produced RAV-O and were sensitive to exogenous RAV-O infection. However, infectious DNA of RAV-O was not obtained from uninfected V- chicken cells or from V+tvbr chicken cells, which spontaneously produced a low titer of RAV-O but were resistant to exogenous RAV-O infection. No detectable amplification of the RAV-O related DNA sequences in the V+tvbs cells was found by hybridization of RAV-O 125I-labeled RNA to the DNAs of V+tvbs and uninfected V- cells. These results indicate that the endogenous avian leukosis virus-related genes in uninfected V- and V+tvbr cells differ from the RAV-O proviruses in RAV-O-infected V- and V+tvbs cells. The lack of infectivity of the DNA of V+tvbr cells is consistent with the hypothesis that the endogenous RAV-O genome in V+tvbr cells is linked to a cis-acting control element, which results in its inefficient expression.