Induction of neoplasms by subgroup E recombinants of exogenous and endogenous avian retroviruses (Rous-associated virus type 60).

Chickens susceptible to infection with subgroup E viruses were inoculated with four independent isolates of Rous-associated virus type 60 (RAV-60) that are subgroup e recombinants of endogenous and exogenous virus. Neoplasms developed in each inoculated group. Therefore, nontransforming viruses of subgroup E can induce lymphoid leukosis at a moderate rate compared with RAV-0, a subgroup E endogenous virus, suggesting that oncogenicity is not a viral envelope (env)-related characteristic. Since the common (c) regions of the RAV-60s examined were of exogenous origin, we suggest that the c region rather than env is important for a high rate of induction of lymphoid leukosis and related neoplasms.     

Intracellular restriction on the growth of induced subgroup E avian type C viruses in chicken cells.

Subgroup E avian type C viruses produced by bromodeoxyuridine-treated 100 X 7, line 7, or line C chicken cells were restricted in their intracellular growth on K28 chicken cells but not on line 15 chicken cells. Cells from embryos of line 15 chickens bred with K28 chickens did not restrict the growth of the subgroup E induced leukosis viruses (ILVs). This result indicates that the phenotype for the intracellular restriction of the growth of subgroup E ILVs found in K28 cells is recessive. Long-term growth of the subgroup E ILVs in K28 cells resulted in the appearance of subgroup E virus that grew well on K28 cells. No change in growth characteristics was observed for subgroup E ILVs grown in line 15 cells indicating that appearance of nonrestricted virus occurred only during growth of the subgrouo E ILVs on a restrictive host. RAV-0, a subgroup E virus closely related to the ilvs, had the same growth characteristics as the subgroup E ILVs. RAV-60, a subgroup E virus formed by recombination of exogenous avian leukosis virus with endogenous subgroup E virus coat information, grew well on both line 15 and K28 cells.     

Component of Strain MC29 Avian Leukosis Virus with the Property of Defectiveness

Three clones of morphologically altered cells (L(-)MC29) of singular properties were isolated from MC29 (subgroup A) leukosis virus-infected chick embryo cells. Supernatant fluids from cultures of the cloned cells produced no transforming or interfering activity on chick embryo cells susceptible to known avian leukosis-sarcoma viruses. No virus associated with the cells was demonstrable by fluorescent-antibody staining or by electron microscopy. All L(-)MC29 clone cells were activated, however, by four strains of Rous-associated viruses (RAV) representative of A, B, C, and D subgroup avian leukosis viruses and by two strains of MC29 virus. Virus L(-)MC29 cells activated by superinfection with RAV-1 and RAV-2 was characterized by helper-dependent and helper-independent properties. These findings suggest that the strain MC29 leukosis virus, or a component thereof, possesses properties of defectiveness similar to those of the Bryan high-titer Rous sarcoma virus.     

Long terminal repeat (LTR) sequences, env, and a region near the 5'' LTR influence the pathogenic potential of recombinants between Rous-associated virus types 0 and 1.

A series of recombinants between Rous-associated virus type 0 (RAV-0), RAV-1, and a replication-competent avian leukosis virus vector (RCAN) have been tested for disease potential in day-old inoculated K28 chicks. RAV-0 is a benign virus, whereas RAV-1 and RCAN induce lymphoma and a low incidence of a variety of other neoplasms. The results of the oncogenicity tests indicate that (i) the long terminal repeat regions of RAV-1 and RCAN play a major role in disease potential, (ii) subgroup A envelope glycoproteins are associated with a two- to fourfold higher incidence of lymphoma than subgroup E glycoproteins, and (iii) certain combinations of 5' viral and env sequences cause osteopetrosis in a highly context-dependent manner. Long terminal repeat and env sequences appeared to influence lymphomogenic potential by determining the extent of bursal infection within the first 2 to 3 weeks of life. This would suggest that bursal but not postbursal stem cells are targets for avian leukosis virus-induced lymphomogenesis. The induction of neutralizing antibody had no obvious influence on the incidence of lymphoma.     

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.     

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.     

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.     

Response to wing-web challenge of Rous sarcoma virus subgroups in some chicken breeds.

Response to wing-web challenge (WWC) of Rous sarcoma virus (RSV) subgroups was studied in 4-8 weeks old chicks of a light breed, a heavy breed and a cross between an indigenous black plumage Bantam fowl and Australorp breed. Wing-web tumor (WWT) began to develop within one week in response to virus subgroups A (BS-RSV) and C [RSV (RAV-49)] challenge. In chicks challenged with subgroup D [RSV (RAV-50)] virus it took a minimum of 4 weeks for development of WWT. Positive response to WWC by subgroups A, C and D virus was 84%, 100% and 52%, respectively. The duration of exhibition of positive response was maximum for subgroup A virus, followed by subgroup D and minimum for subgroup C virus.     

Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus.

Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing from those of either parental virus. Recombinants carrying the gag-pol region of RPV and the env gene of UR2AV induced a high incidence of a muscle lesion termed infiltrative intramuscular fibromatosis. One recombinant, EU-8, which carries the gag-pol and LTR sequences of RPV, and the env gene of UR2AV, induced lymphoid leukosis after an unusually short latent period. The median time of death from lymphoid leukosis was 6 to 7 weeks after infection with EU-8 compared with approximately 5 months for UR2AV.     

Recombinants between endogenous and exogenous avian tumor viruses: role of the C region and other portions of the genome in the control of replication and transformation.

Endogenous retroviruses of chickens are closely related to exogenous viruses isolated from spontaneous tumors in the same species, yet differ in a number of important characteristics, including the ability to transform cells in culture, ability to cause sarcomas or leukemias, host range, and growth rate in cell culture. To correlate these differences with specific sequence differences between the two viral genomes, the genome RNA of transforming subgroup E recombinants between the Prague strain of Rous sarcoma virus, subgroup B (Pr-RSV-B), and the endogenous Rous-associated virus-0 (RAV-0), Subgroup E, and seven nontransforming subgroup E recombinants between the transformation-defective mutant of Pr-RSV-B and RAV-0 was examined by oligonucleotide fingerprinting. The pattern of inheritance among the recombinant viruses of regions of the genome in which Pr-RSV-B and RAV-0 differ allowed us to draw the following conclusions. (i) Nonselected parts of the genome were, with a few exceptions, inherited by the recombinant virus progeny randomly from either parent, with no obvious linkage between neighboring sequences. (ii) A small region in the Pr-RSV-B genome which maps in the 5' region was found in all transforming but only some of the nontransforming recombinants, suggesting that it plays a role in the control of the expression of transformation. (iii) A region of the Pr-RSV-B genome which maps between env and src was similarly linked to the src gene and may be either part of the structural gene for src or a control sequence regulating the expression of src. (iv) The C region at the extreme 3' end of the virus genome which is closely related in all the exogenous avian retroviruses but distinctly different in the endogenous viruses is the major determinant responsible for the differences in growth rate between RAV-0 and Pr-RSV-B. This latter observation allowed us to redefine the C region as a genetic locus, c, with two alleles cn (in RAV-0) and cx (in exogenous viruses).     

Sequence comparison in the crossover region of an oncogenic avian retrovirus recombinant and its nononcogenic parent: genetic regions that control growth rate and oncogenic potential.

NTRE 7 is an avian retrovirus recombinant of the endogenous nononcogenic Rous-associated virus-0 (RAV-0) and the oncogenic, exogenous, transformation-defective (td) Prague strain of Rous sarcoma virus B (td-PrRSV-B). Oligonucleotide mapping had shown that the recombinant virus is indistinguishable from its RAV-0 parent except for the 3'-end sequences, which were derived from td-PrRSV-B. However, the virus exhibits properties which are typical of an exogenous virus: it grows to high titers in tissue culture, and it is oncogenic in vivo. To accurately define the genetic region responsible for these properties, we determined the nucleotide sequences of the recombinant and its RAV-0 parent by using molecular clones of their DNA. These were compared with sequences already available for PrRSV-C, a virus closely related to the exogenous parent td-PrRSV-B. The results suggested that the crossover event which generated NTRE 7 took place in a region -501 to -401 nucleotides from the 3' end of the td-PrRSV parental genome and that sequences to the right of the recombination region were responsible for its growth properties and oncogenic potential. These sequences included a 148-base-pair exogenous-virus-specific region that was absent from the RAV-0 genome and the U3 region of the long terminal repeat. Since the exogenous-virus-specific sequences are expected to be missing from transformation-defective mutants of the Schmidt-Ruppin strain of RSV, which, like other exogenous viruses, grow to high titers in tissue culture and are oncogenic in vivo, we concluded that the growth properties and oncogenic potential of the exogenous viruses are determined by sequences in the U3 region of the long terminal repeat. However, we propose that the exogenous-virus-specific region may play a role in determining the oncogenic spectrum of a given oncogenic virus.     

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.     

The effect of avian retroviruses on limb bud chondrogenesis in vitro

Mesenchymal cells isolated from stage 24 embryonic chicken limb buds were infected with the temperature-sensitive transformation mutants of Rous sarcoma virus tsNY68, tsNY10 and tsLA25 at the nonpermissive temperature for transformation (41 degrees C). Virus infection greatly inhibited subsequent limb bud chondrogenesis under nontransforming conditions, as indicated by a reduction in the rate of 35SO4 incorporation into cell-associated proteoglycans. The inhibition of chondrogenesis was directly related to the percentage of cells infected with tsNY68 at 41 degrees C. The observed inhibition of chondrogenesis was independent of src gene expression since this effect was also caused by many viruses which lack the src gene, including the leukosis viruses RAV-1, RAV-2 and MAV-2(0); the src deletion mutant RSVtd107; and the reticuloendotheliosis viruses REV-T and SNV. Infection of mesenchymal cells with tsNY68 under nontransforming conditions did not cause changes in parameters such as the rate of thymidine incorporation, total cell DNA and total cell protein. Infection with tsNY68 at 41 degrees C resulted in altered kinetics of 35SO4 incorporation into cell-associated proteoglycans and a corresponding reduction in 35SO4-labeled proteoglycans extracted from the cell layer. There were no apparent quantitative effects on the rate of accumulation of proteoglycans in the culture medium. The proteoglycans extracted from the cells and the collected medium of tsNY68-infected cultures were smaller than those of uninfected cultures, as shown by agarose gel chromatography.     

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.     

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.     

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.     

Development of avian sarcoma and leukosis virus-based vector-packaging cell lines.

We have constructed an avian leukosis virus derivative with a 5' deletion extending from within the tRNA primer binding site to a SacI site in the leader region. Our aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1 psi-, to develop vector-packaging cell lines. We show that RAV-1 psi- can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, we have demonstrated that QT6-derived lines expressing RAV-1 psi- can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-1 psi--expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. We show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.     

Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas.

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNA-RNA hybridization under conditions of DNA excess. Comparisons were made between 60-70S RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of "laboratory" leukosis and sarcoma viruses, by competitive hybridization analysis. A minimum of 18% of the genome sequences of both ALV isolates detected in DNA from lymphomas they induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken geome showed significant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The increased hybridization of ALV RNA with lymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabricius following infection.increased hybridization was correlated with both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation.     

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.