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Several clones of Fujinami sarcoma virus (FSV) isolated from a laboratory stock or from mutagenized virus were temperature sensitive (ts) in transformation of cells in culture. When shifted from the permissive (37 degrees C) to the nonpermissive (41.5 degrees C) temperature, the cellular phenotype reverted to normal within 2 h, but it required about 48 h at 37 degrees C to revert back to the transformed morphology. A temperature-resistant (tr) FSV clone was isolated from a tumor of an animal. All ts mutants were tumorigenic in animals but induced tumors only after latent periods of 12 to 25 days, compared to 5 to 6 days with tr virus. The ts lesions of the FSV mutants affected 90% of the phosphorylation of the nonstructural, gag-related 140,000-kilodalton phosphoprotein coded by FSV (p140), but did not affect virus replication or the synthesis of p140. Upon shifting from the permissive to the nonpermissive temperature, p140 was 90% dephosphorylated with an approximate (32)P half-life of 20 min. When shifted back to the permissive temperature, the preexisting p140 was rephosphorylated in the absence of protein synthesis within a 90-min test period. Likewise, most of the phosphate of fully phosphorylated p140 was exchanged at the permissive temperature within 30 to 90 min even when protein synthesis was inhibited. However, the protein structure of p140 had a half-life of 5 h at both temperatures. These results prove p140 to be a substrate of reversible phosphorylation. Superinfection and transformation of ts FSV-infected cells maintained at the nonpermissive temperature with acute leukemia virus MC29 failed to phosphorylate p140. It would follow that in vivo phosphorylation of ts p140 is controlled by an FSV-specific mechanism and is a prerequisite, not a consequence, of transformation. p140 of ts FSV recovered from cells maintained at 41.5 degrees C with anti-gag serum was over 10 times less phosphorylated by associated kinase than the same protein recovered from cells at 37 degrees C if assayed in vitro at 20 degrees C. This kinase activity associated with or dissociated from p140 with a half-life of less than 30 min during temperature shifts of ts FSV-infected cells. However, p140 recovered from ts FSV-infected cells maintained at 37 degrees C was phosphorylated by associated kinase in vitro not only at 20 degrees C but also, and essentially at the same level, at 41.5 degrees C. This suggests that the kinase associated with the immunocomplex of p140 of ts FSV is not temperature sensitive. p140 translated in vitro from ts and tr FSV RNA lacked kinase activity. We conclude that a fully phosphorylated p140 is necessary for the maintenance of transformation by FSV. This is consistent with the notion that other highly oncogenic viruses also code for nonstructural phosphoproteins with probable transforming function. A model which postulates that p140 is a substrate of reversible phosphorylation and that the lesion of the ts FSV clones described herein affects association of p140 with a cellular kinase rather than a hypothetical intrinsic kinase activity of the protein is most compatible with our data.

The response to infection of chicken hemopoietic cells derived from the early stages of embryogenesis by avian myeloblastosis virus (AMV) and avian erythroblastosis virus (AEV) was investigated. It was found that erythroid progenitor cells were present in the blastoderm at a higher frequency than that of myeloid progenitor cells. These results correlate with the observation that target cells for AEV were found to be more numerous than those for AMV. Therefore, blastoderm cells are of potential value in understanding the mechanisms of oncogenesis at the level of the target cells.

The article presents the data obtained by the authors in studies of virus-viral co-cancerogenesis, the interaction between some non-oncogenic viruses and well-known oncogenic viruses, the results of co-cancerogenic effect Marek's disease herpesvirus with the avian leukemia virus and the possibility of phenotype mixing between oncornaviruses belonging to different species in nature.

The Fujinami avian sarcoma virus (FSV) transforming gene product, P140, is a fusion protein which contains both gag-related and FSV-specific methionine-containing tryptic peptides. The virion protease p15 cleaved p140 into two fragments: an N-terminal 33K fragment which contained all but one of the gag-related tryptic peptides and a C-terminal 120K fragment which contained all of the FSV-specific tryptic peptides. The 33K gag-related fragment from P140 phosphorylated in FSV-transformed cells contained only phosphoserine, whereas the 120K C-terminal FSV-specific fragments contained both phosphoserine and phosphotyrosine. P140 isolated from cells infected at the nonpermissive temperature with an isolate of FSV which is temperature sensitive for transformation had a normally phosphorylated 33K fragment, but a hypophosphorylated 120K fragment deficient in both phosphotyrosine and phosphoserine. When P140 was immunoprecipitated from cells and phosphorylated in vitro at tyrosine residues in the immune complex kinase reaction, only the FSV-specific fragment was labeled. These data define the structure of FSV P140 and locate the phosphorylated amino acids within the two regions of the polypeptide.

The transformed or normal phenotype of cultured normal rat kidney cells infected with a temperature-sensitive mutant of avian sarcoma virus is conditional on the temperature at which the cells are grown. Using dye injection techniques, we show that junction-mediated dye transfer is also temperature-sensitive. The extent and rate of transfer between infected cells grown at the transformation-permissive temperature (35 degrees C) is significantly reduced when compared to infected cells grown at the nonpermissive temperature (40.5 degrees C) or uninfected cells grown at either temperature. Infected cells subjected to reciprocal temperature shifts express rapid and reversible alterations of dye transfer capacities, with responses evident by 15 min and completed by 60 min for temperature shifts in either direction. These results suggest that altered junctional capacities may be fundamental to the expression of the ASV-induced, transformed phenotype.

Cultures of established rat fibroblasts transformed by the avian erythroblastosis virus were more susceptible to the cytopathic effect of the autonomous parvovirus minute virus of mice, prototype strain (MVMp), than were their untransformed homologs. This effect could be ascribed to the presence of a greater fraction of cells that were sensitive to the killing action of MVMp in transformed cultures than in their normal parents. Yet, transformed and normal lines were similarly efficient in virus uptake, DNA amplification, and capsid protein synthesis. In contrast, transformants accumulated 2.5- to 3-fold greater amounts of all three major MVM mRNA species and nonstructural protein than did their normal progenitors. Thus, in this system transformation-associated sensitization of cells to MVMp appears to correlate primarily with an increase in their capacity for the expression of the viral transcription unit which encodes nonstructural proteins and is controlled by the P4 promoter. Consistently, a reporter gene was expressed at a higher level by transformed versus normal cultures, when placed under the control of the MVM P4 promoter. As infectious MVMp was produced in larger amounts by transformed cultures, a late step of the parvoviral cycle, such as synthesis, encapsidation of progeny DNA, or both, was also stimulated in the transformed cells.

The H strain of avian erythroblastosis virus (AEV-H) was recently isolated from the liver emulsion of a chicken that suffered from erythroblastosis after being inoculated with subgroup A leukosis virus. AEV-H induced erythroblastosis or sarcoma when inoculated into chickens and transformed chick embryo fibroblasts (CEF) in vitro. Analysis of viral proteins synthesized in cells, which were named HNP, transformed by AEV-H but not producing transforming virus revealed tha the genome of AEV-H directed the synthesis of the gag gene products, Pr76gag and Pr180gag-pol, which was the precursor of active reverse transcriptase. Thus the HNP produced virions that were not infectious due to a defect of the env gene. Studies on the viral RNA showed that a 35 S RNA, estimated to be 8000 nucleotides long, was the genomic RNA of AEV-H and probably carried one transforming gene, which is most likely erbB gene. The gene organization of AEV-H was suggested to be 5'-gag-pol-onc-3'. These data imply that the single oncogene is responsible for both erythroblastosis and sarcoma.

We previously reported the isolation of a newly recovered avian sarcoma virus (rASV) from tumors of chickens injected with transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV). In this paper, we present further biological and biochemical characterization of the recovered sarcoma viruses. High titers of rASV's were generally obtained by cocultivation of tumor cells with normal chicken embryo fibroblasts or by homogenization of tumor tissues. Most rASV isolates were similar to SR-RSV, subgroup A (SR-RSV-A), in their growth characteristics and were nondefective in replication. The subgroup specificity of rASV's and the electrophoretic mobilities of their structural proteins were the same as those parental td viruses. The nondefectiveness of rASV's was further substantiated by the size of their genomic RNA, which was indistinguishable from that of SR-RSV-A and substantially larger than that of parental td RNA. Molecular hybridization using complementary DNA specific to the src gene of SR-RSV (cDNAsrc) showed that the RNAs of td mutants used in this study contained extensive deletions within the src gene (7 to 30% hybridization with cDNAsrc); the same probe hybridized up to 90% with RNA from two isolates of rASV. These data indicate that rASV has regained genetic information which had been deleted in the td mutants and strongly suggest that the generation of rASV involves a genetic interaction between td virus and host cell genetic information.

The OK 10 virus complex was isolated from a liver tumour of a chicken which, as an embryo, had been inoculated intravenously with a field isolate of an avian leukosis virus. The OK 10 virus complex contains at least two viruses: the interference assay and serum neutralization test indicate that the helper virus belongs to subgroup A. One of the viruses, OK 10 V, induces distinct foci in chick embryo cells under agar overlay and cells from the foci form colonies in soft agar. These properties allow in vitro assay of the virus. Injection of virus or infected cells into chicks induces acute leukaemia but no local tumours. Another virus, OK 10 AV (associated virus), comprises about 99% of the OK 10 complex. The virus does not induce foci in chick embryo cells. In chickens it causes leukosis 17 months after injection. Electron micrographs of OK 10 virus stocks show typical C type virus particles. These particles have a density of 1.16 g/ml and contain 70S RNA which, after heat denaturation, releases type b RNA subunits. The OK 10 virus complex apparently represents a strain of acute leukaemia viruses.

We studied the effects of dexamethasone on virus production and antigen expression in avian retrovirus-infected chick embryo fibroblast (CEF) cells. The presence of specific receptors for this hormone in both normal and infected CEF cells could only be demonstrated using cultures that were at or near confluence. Dexamethasone, while not toxic to the cells, exerted a growth-retarding influence when employed at concentrations of 10(-8) or 10(-9) M. These same concentrations of hormone were inhibitory to virus production, elaboration of plasminogen activator activity, and antigen expression as determined in a sensitized lymphocyte stimulation assay. In contrast, infected cells that had been treated with hormone displayed enhanced reactivity in a specific immunofluorescence test.

The new avian retroviruses UR1 and UR2 were isolated from spontaneous tumors of chickens by cocultivation of tumor material with susceptible chicken embryo fibroblasts. In vitro, UR1 induced formation of small foci of round and fusiform cells. On the other hand, cells infected by UR2 assumed an extremely elongated morphology. In vivo, both viruses induced fibrosarcomas and myxosarcomas with short latencies. Infectivity assays with and without mitomycin C showed that both viruses were defective for replication, but transformed nonproducing cell clones were obtained only with UR1. UR1-infected transformed nonproducing clones did not release particles detectable by reverse transcriptase assays, and fusion of transformed nonproducing cells with quail cells chronically infected with Rous sarcoma virus (a Bryan strain) failed to rescue infectious virus. This suggested that UR1 does not code for functional envelope glycoproteins. In this regard, UR1 appeared to be similar to Fujinami, PRCII, and Y73 viruses. The helper viruses of partially purified stocks of UR1 and UR2 appeared to belong to subgroup A, but these helper viruses were distinguishable from each other, as shown by host range experiments and neutralization tests. Hybridization studies with DNA complementary to the src gene of Rous sarcoma virus and RNAs extracted from both UR1 and UR2 showed no homology between the genomes of the new isolates and the transforming gene of Rous sarcoma virus.

A continuous chicken erythroblast cell line transformed by the temperature-sensitive mutant ts34 of avian erythroblastosis virus was developed. This cell line, designated HD3, could be induced to terminally differentiate by shift to the nonpermissive temperature. The differentiated cells resembled erythrocytes as judged by morphology, expression of hemoglobin as determined by benzidine staining and radioimmunoassay, and by the expression of differentiation-specific cell surface antigens. Terminal differentiation was dependent on an erythropoietin-like activity present in anemic chicken serum. In contrast, induction of differentiation in the same cells by butyric acid was erythropoietin independent and did not lead to the formation of erythrocytes. In addition, we found that the responsiveness to temperature inducibility and to butyric acid could be dissociated in variant sublines of HD3 and that both types of differentiation inducers appear to act via different pathways.

RSV transforms several nonhematopoietic cell types and as reported here also has the capacity to transform hematopoietic cells of the erythroid lineage. In vitro, the three RSV isolates tested induced erythroblast-like colonies in infected bone marrow cells that were distinguishable by size and cell arrangement from those induced by avian erythroblastosis virus (AEV). Also in contrast to AEV-transformed erythroblast cultures, isolated cell colonies induced by RSV required complex growth conditions in liquid medium similar to the in vitro conditions necessary for erythroblasts transformed by the acute leukemia virus E26. Temperature-shift experiments using temperature-sensitive (ts) NY68 RSV revealed that when grown at the nonpermissive temperature (42 degrees), mutant-infected cells became benzidine positive and partially differentiated into erythrocytes. Wild-type (wt) RSV-transformed cells did not undergo similar changes. However, both wt RSV-, and to a greater extent, ts RSV-transformed cultures at the permissive temperature (37 degrees) did contain populations of spontaneously differentiating erythroid cells signifying that the transforming activity of the virus did not fully arrest erythroid maturation. In addition, the RSV-transformed cells did express tyrosine kinase activity. When injected intravenously into birds, RSV induced an erythroblastosis-like disease similar to AEV but also caused fibrosarcomas and leg paralysis. These results show that RSV can alter the pattern of erythroid differentiation in a manner similar to, but distinct from, AEV and indicate that the tyrosine-specific pp60src kinase is involved in erythroid cell transformation. Since the src and erb B proteins share a significant amino acid homology, these data suggest that both may also share a common functional homology.

Avian erythroblastosis virus (AEV) induces erythroblastosis and fibrosarcomas. The viral erbB protein is required for AEV-mediated oncogenesis. To explore the structural aspects of the v-erbB polypeptide necessary for its oncogenic function, we created a series of small in-frame insertions in different domains of the v-erbB oncogene. AEV genomes bearing lesions within the v-erbB kinase domain demonstrated a drastically decreased ability to transform avian fibroblasts, establishing a functional role for this structurally conserved oncogene domain. In contrast, mutations in the extracellular domain, between the transmembrane region and the kinase domain, or at the extreme C terminus of the v-erbB protein had no effect on AEV-mediated fibroblast transformation. One lesion within the v-erbB kinase domain, a 10-amino acid insertion, produced a temperature-sensitive mutant capable of fibroblast transformation at 36 degrees C but not at 41 degrees C, suggesting that small in-frame insertions have general utility for the in vitro creation of conditional mutants.

For the first time, we present evidence with restriction enzymes HpaII and MspI which indicates that the proviral DNA sequence of avian sarcoma virus is modified by methylation in a nonpermissive rat cell line but not in permissive chicken cells. Some of the endogenous viral sequences in the permissive cells were also methylated. No 5-methylcytosine could be detected in the unintegrated viral DNA.

Neoplastic transformation of cell by avian sarcoma virus is mediated by a single viral gene (src), which encodes a phosphoprotein (pp60src) with the enzymatic activity of a protein kinase. The DNAs of vertebrate species contain a highly conserved homologue of src that is also represented in the polysomal RNA of uninfected cells and, hence, may specify a normal cellular protein. We have used antisera directed against pp60src to isolate a closely related phosphoprotein (denoted vertebrate pp60) from uninfected chicken, quail, rat, and human cells. Our data indicate that vertebrate pp60 is a homologue of pp60src, highly conserved both antigenically and chemically. Moreover, the cellular protein may possess protein kinase activity similar to that associated with pp60src. We conclude that the product of src is a slightly modified analogue of a normal cellular protein.

The avian leukemia sarcoma virus S13 transforms chicken and Japanese quail embryo fibroblasts and chicken erythroid cells in tissue culture. S13-induced erythroid transformation requires culture conditions suitable for the growth of normal erythroid precursors (H. Beug and M. J. Hayman (1984), Cell 36, 963-972). S13-transformed erythroid colonies contain a high percentage of cells that differentiate in absence of erythropoietin. S13 is defective in pol and env functions but can code for a complete set of gag proteins. Nonproducer cell clones transformed by S13 release a noninfectious viral particle containing gag but no functional env or pol proteins. They also synthesize a transformation-specific protein of 155,000 molecular weight. This protein reacts with antibody to viral envelope glycoproteins and appears to represent onc as well as env sequences. The 155,000-molecular weight env-linked protein does not cross react immunologically with an antiserum against the v-erb A and v-erb B gene products.