gag as well as myc sequences contribute to the transforming phenotype of the avian retrovirus FH3.

The avian retrovirus FH3, like MC29 and CMII, encodes a Gag-Myc fusion protein. However, the FH3-encoded protein is larger, about 145 kDa, and contains almost the entire retroviral gag gene. In contrast to the other gag-myc avian retroviruses, FH3 fails to transform fibroblasts in vitro, although macrophages are transformed both in vitro and in vivo (C. Chen, B. J. Biegalke, R. N. Eisenman, and M. L. Linial, J. Virol. 63:5092-5100, 1989). We have used the polymerase chain reaction technique to obtain a molecular clone of FH3. Sequence analysis of the FH3 myc oncogene revealed a single proline----histidine change (position 223) relative to c-myc. However, substitution of the FH3 myc sequence with the chicken c-myc sequence did not alter the transformation potential of the virus. Hence, overexpression of the proto-oncogene as a Gag-Myc retroviral protein is sufficient for macrophage, but not fibroblast, transformation. After passage of FH3 in fibroblast cultures, a virus (FH3L) that is capable of rapidly transforming fibroblasts appears. The Gag-Myc protein encoded by FH3L is smaller (ca. 130 kDa) than that encoded by the original viral stock (FH3E). Sequencing of an FH3L molecular clone revealed a 212-amino-acid deletion within the Gag portion. Using FH3E/FH3L recombinants, we have demonstrated that the ability of encoded viruses to transform fibroblasts directly correlates with the presence of this deletion. Moreover, the addition of the Gag sequence deleted from FH3L to the MC29 oncoprotein significantly reduces its transforming activity as measured by focus assay. These data suggest that the C-terminal segment of Gag attenuates the oncogenic potential of Gag-Myc fusion proteins.     

Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location.     

Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

Generation of transforming viruses in cultures of chicken fibroblasts infected with an avian leukosis virus.

During serial passages of an avian leukosis virus (the transformation-defective, src deletion mutant of Bratislava 77 avian sarcoma virus, designated tdB77) in chicken embryo fibroblasts, viruses which transformed chicken embryo fibroblasts in vitro emerged. Chicken embryo fibroblasts infected with these viruses (SK770 and Sk780) had a distinctive morphology, formed foci in monolayer cultures, and grew independent of anchorage in semisolid agar. Bone marrow cells were not transformed by these viruses. Another virus (SK790) with similar properties emerged during serial subcultures of chicken embryo fibroblasts after a single infection with tdB77. The 50S to RNAs isolated from these viruses contained a tdB77-sized genome (7.6 kilobases), 8.7- and 5.7-kilobase RNAs, and either a 4.1-kilobase RNA or a 4.6-kilobase RNA. These RNAs did not hybridize with cDNA's representing the src, erb, mac, and myb genes of avian acute transforming viruses. Cells transformed by any one of the Sk viruses (SK770, SK780, or SK790) synthesized two novel gag-related polyproteins having molecular weights of 110,000 (p110) and 125,000 (p125). We investigated the compositions of these proteins with monospecific antiviral protein sera. We found that p110 was a gag-pol fusion protein which contained antigenic determinants, leaving 49,000 daltons which was antigenically unrelated to the structural and replicative proteins of avian leukosis viruses. An analysis of the SK viral RNAs with specific DNA probes indicated that the 5.7-kilobase RNA contained gag sequences but lacked pol sequences and, therefore, probably encoded p125. The transforming ability, the deleted genome, and the induced polyproteins of the SK viruses were reminiscent of the properties of several replication-defective acute transforming viruses.     

Replicating, differentiated macrophages can serve as in vitro targets for transformation by avian myeloblastosis virus.

Pure cultures of chicken macrophages were characterized functionally and transformed by avian myeloblastosis virus. Transformed cells exhibited an altered function. The efficiency of transformation was limited by the mitotic activity of the macrophages.     

Subcellular localization of proteins encoded by oncogenes of avian myeloblastosis virus and avian leukemia virus E26 and by chicken c-myb gene

Analysis of the subcellular location of the proteins encoded by the oncogenes of avian myeloblastosis virus and avian leukemia virus E26 ( p45v -myb and p135gag -myb-ets, respectively) and by the chicken c-myb gene ( p75c -myb) shows that all three proteins are located in the nucleus. In AMV-infected (but not transformed) chicken fibroblasts p45v -myb also resides in the nucleus, indicating that a nuclear location of p45v -myb in these cells is not sufficient to achieve transformation. In AMV-transformed myeloblasts a small fraction of p45v -myb occupies an additional site in the perinuclear region of the cytoplasm. If the myeloblasts are caused to differentiate to macrophages, most of p45v -myb is found in the cytoplasm. This redistribution of p45v -myb within the cell may be responsible for reversion of the transformed phenotype.     

Avian myeloblastosis provirus cloned in a lambda bacteriophage is leukemogenic

The avian myeloblastosis virus provirus inserted in a lambda bacteriophage, recombinant clone 11A1-1 (Souza et al., Proc. Natl. Acad. Sci. U.S.A. 77:3004-3008, 1980), was transfected into chicken embryo fibroblasts which had been preinfected with either Rous-associated virus type 61 or the transformation-defective avian sarcoma virus tdB77. Within 4 to 5 h after transfection, the cells were injected into 16-day-old chicken embryos or 1-day-old chicks. Acute myeloblastic leukemia developed after a long latent period. Filtered (0.22-micrometer pores) supernatant of transformed buffy-coat cell cultures from one leukemic chicken of the lambda 11A1-1 (tdB77) group rapidly transformed yolk sac cells in vitro. Results from an infectivity interference assay and analysis of proviral DNA fragments generated with restriction endonucleases were consistent with the presence in leukemic cells of defective avian myeloblastosis virus and tdB77 as the helper virus.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector.

The middle T antigen of polyomavirus transformed primary chicken embryo fibroblasts when expressed from a replication-competent avian retrovirus. This in vitro-constructed retrovirus, SRMT1, is a variant of Rous sarcoma virus that encodes the middle T antigen in place of v-src. Inoculation of SRMT1 into 1-week-old chickens rapidly induced hemangiomas and hemangiosarcomas. As shown with mammalian cells infected with polyomavirus, polyomavirus middle T antigen appears to be associated with p60c-src in chicken cells infected with SRMT1. When lysates of SRMT1-infected cells immunoprecipitated with either a monoclonal antibody against p60src or anti-T serum were assayed in an in vitro kinase reaction, the middle T antigen was heavily phosphorylated. To see whether an excess of p60c-src could alter the extent of phosphorylation of the middle T protein or the process of cell transformation by middle T, cells were doubly infected with SRMT1 and NY501, a virus which overexpresses p60c-src. Doubly infected chicken embryo fibroblasts transformed with the same kinetics and were morphologically indistinguishable from chicken embryo fibroblasts infected with SRMT1 alone. Phosphorylation of the middle T antigen was elevated two- to fivefold relative to cells infected only with SRMT1.     

Expression of Embryonic Haemoglobin in ts AEV-Transformed Embryonic Erythroid Cells During Temperature-Induced Differentiation

Cells prepared from 1-day-old chick blastoderms were infected with a temperature-sensitive mutant of avian erythroblastosis virus ( ts AEV). Clonal strains of transformed erythroblasts were isolated from the infected blastoderm cells. By shift to the nonpermissive temperature, these cells could be induced to differentiate into erythrocyte-like cells which expressed embryonic haemoglobins. Embryonic haemoglobins could not be detected in ts AEV-transformed erythroblasts from adult bone marrow when induced to differentiate under the same conditions. In contrast to normal primitive erythrocytes, ts AEV-infected embryonic erythroblasts differentiated in vitro expressed also adult haemoglobin. These results suggest an influence of the haematopoietic environment on the switch from embryonic to adult erythrocytes.     

Assay of noninfectious fragments of DNA of avian leukosis virus-infected cells by marker rescue.

A marker rescue assay of noninfectious fragments of avian leukosis virus DNAs is describe. DNA fragments were prepared either by sonication of EcoRI-digestion of DNAs of chicken cells infected with wild-type Rous sarcoma virus, with a nontransforming avian leukosis virus, and with a mutant of Rous sarcoma virus temperature sensitive for transformation. Recipient cultures of chicken embryo fibroblasts were treated with noninfectious DNA fragments and infected with temperature-sensitive mutants of Rous sarcoma virus defective in DNA polymerase or in an internal virion structural protein. Wild-type progeny viruses which replicated at the nonpermissive temperature were isolated. Some of the wild-type progeny acquired both the wild-type DNA polymerase and the subgroup specificity of the Rous sarcona virus strain used for preparation of sonicated or EcoRI-digested DNA fragments. Therefore the genetic markers for DNA polymerase and envelope were linked and appeared to be located on the same EcoRi fragment of the DNA of Rous sarcoma virus-infected cells.     

Transfection of Chicken Embryo Cells with DNA Extracted from Avian Virus-Producing Neoplastic Cells

DNA isolated from avian virus-producing leukemic myeloblasts induced the production of viruses, but not morphological transformation, in cultivated chicken fibroblasts. The recovered virus had the same biological characteristics as the original avian myeloblastosis virus (AMV) and produced myeloblastosis and nephroblastomas when injected into chickens. Neutralization experiments with chicken anti-AMV-BAI strain A sera showed an antigenic community between the DNA-transfected virus and the original virus. Virus induced in fibroblasts after treatment with DNA from a viral nephroblastic nephroblastoma line only gave nephroblastoma when injected into chicken. Treatment of chicken embryo cells with DNA extracted from normal chicken embryos did not induce viral production.     

Molecular weight dependence of hyaluronic acid produced during oncogenic transformation

Cultured chicken embryo fibroblasts synthesize two distinct molecular size classes of hyaluronic acid. The high molecular weight material (form I, 2.98 x 10(6) is the predominant species synthesized by transformed cells, whereas form II (1.42 x 10(5)) is the major product of non-transformed cells. A shift to synthesis of predominantly form I hyaluronic acid is an early transformation event in cells infected with LA24 Rous sarcoma virus and maintained at the permissive temperature for transformation (35 degrees C). Form I hyaluronic acid exhibits greater binding to preparations of cellular fibronectin and to both normal and transformed cells than does form II. Both forms bind more to transformed cells than to normal, uninfected cells. Hyaluronic acid (predominantly form I) isolated from transforming cells stimulates proliferation in growth-retarded, non-transformed cells.     

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.     

Chicken macrophages synthesize and secrete avidin in culture

It was previously shown that avidin, a glycoprotein secreted in vivo by chicken oviduct, is produced by cultured transformed or damaged chicken embryo fibroblasts [27]. This report demonstrates synthesis and secretion of large amounts of avidin by macrophages isolated from chicken yolk sac. Avidin was secreted to the culture medium as shown by immunoprecipitation of metabolically labeled proteins. In the culture medium of macrophages the avidin concentration (up to 47.5 +/- 0.5 microgram/mg cellular protein) exceeded, in agreement with previous findings, that of fibroblasts (up to 7.3 +/- 0.7 microgram/mg) infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature sensitive for transformation and OK 10 virus). No difference between the macrophage avidin and the egg white avidin was detected by both the heat-induced [14C] biotin exchange assay and immunoblotting (subunit Mr = 15600). By immunofluorescence 10 to 20% of the cells were positive for avidin, independent of the time in culture (1-30 days). The staining pattern varied between dense or granular perinuclear and strong reticulo-granular fluorescence throughout the cytoplasm. Double staining for avidin and the Golgi region by wheat germ agglutinin showed that avidin is concentrated, and might be processed, in the Golgi complex. The production of avidin by macrophages supports a role for avidin in host defence mechanisms.     

Cell Cycle-Dependent Activation of Rous Sarcoma Virus-Infected Stationary Chicken Cells: Avian Leukosis Virus Group-Specific Antigens and Ribonucleic Acid

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary cells did not contain avian leukosis virus group-specific antigens or ribonucleic acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV endogenous RNA-directed DNA polymerase activity.     

Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses.

The level of phosphotyrosine in vinculin was determined in chicken embryo fibroblasts transformed by various strains of avian sarcoma virus. As previously reported (Sefton et al., Cell 24:165-174, 1981), vinculin was phosphorylated at tyrosine residues in most cultures examined, but the level varied greatly and no detectable change was found in cultures infected with Fujinami sarcoma virus or UR2 sarcoma virus. Regardless of the level of vinculin phosphorylation, the number of organized microfilament bundles was found to be decreased in all transformed cells. These results strongly suggest that tyrosine phosphorylation of vinculin is not an obligatory step in cell transformation by this class of oncogenes, nor is it correlated with the associated cytoskeletal disarray.     

Transformation of mammalian fibroblasts and macrophages in vitro by a murine retrovirus encoding an avian v-myc oncogene

A murine retrovirus which expresses the avain v-myc OK10 oncogene was constructed. The virus, denoted MMCV, readily transforms fibroblasts of established lines, such as mouse NIH/3T3 and rat 208F cells, to anchorage-independent growth in agarose. The virus also transforms primary mouse cells: (i) virus-infected macrophages are induced to form large colonies in semi-solid media, and can easily be expanded into mass cultures; (ii) MMCV-infected fibroblastic cells from mouse limb buds undergo morphological transformation and grow in semi-solid medium. MMCV thus transforms both mouse fibroblastic cells and macrophages in vitro, in a fashion similar to the v-myc-containing avian viruses in chicken cells. The possibility of introducing a transforming myc gene into mammalian cells by virus infection provides a novel approach for studying the mechanism of myc transformation in cells from many lineages.     

Spontaneous Conversion of Nontransformed Avian Sarcoma Virus-Infected Rat Cells to the Transformed Phenotype

Normal rat kidney (NRK) fibroblasts were infected with the Schmidt-Ruppin strain (SR-D) of avian sarcoma virus (ASV) and cloned 20 h after infection without selection for the transformed phenotype. Most infected clones initially exhibited the flat, nontransformed morphology that is characteristic of uninfected NRK cells. In long-term culture, however, the majority of the SR-D NRK clones began segregating typical ASV-transformed cells. Transforming ASV could be rescued by fusion with chicken embryo fibroblasts from most of the infected clones tested. Three predominantly flat, independently infected clones were further analyzed by subcloning 8 to 10 weeks after infection. Most flat progeny subclones derived at random from two of these "parental" SR-D NRK clonal lines did not yield virus upon fusion with chicken embryo fibroblasts, although a nondefective transforming ASV was repeatedly recovered from the parental clones. This observation suggested that most, but not all, daughter cells in these SR-D NRK clones lost the ASV provirus after cloning. The progeny of the third independent parental cell clone, c17, gave rise to both flat and transformed subclones that carried ASV. In this case, ASV recovery by fusion and transfection from the progeny subclones was equally efficient regardless of the transformation phenotype of the cells. The 60,000-dalton phosphoprotein product of the ASV src gene was, however, expressed at high level only in the transformed variants. The results of a Luria-Delbruck fluctuation analysis and of Newcombe's respreading test indicated that the event leading to the spontaneous conversion to the transformed state occurred at random in dividing cultures of these flat ASV NRK cells at a rate predicted for somatic mutation.