Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.

We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA.

Coordinate regulation of myelomonocytic phenotype by v-myb and v-myc.

Both avian myeloblastosis virus (by the action of v-myb) and avian myelocytomatosis virus MC29 (by the action of v-myc) transform cells of the myelomonocytic lineage. Whereas avian myeloblastosis virus elicits a relatively immature phenotype, cells transformed by MC29 resemble mature macrophages. When cells previously transformed by v-myb were superinfected with MC29, their phenotype was rapidly altered to that of a more mature cell. These superinfected cells expressed both v-myb (at a level similar to that found before superinfection) and v-myc. It therefore appears that the expression of v-myc can elicit certain properties of a more differentiated phenotype. In addition, unlike cells transformed by v-myb alone, the cells expressing both v-myb and v-myc could not be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to differentiate to fully mature macrophages. Cells with a morphology similar to that of the superinfected cells were elicited by simultaneously infecting yolk sac macrophages with avian myeloblastosis virus and MC29. Such cells expressed both v-myb and v-myc. These results indicate that expression of v-myb and v-myc in infected cells coordinately regulates myelomonocytic phenotype and that the two viral oncogenes vary in their ability to interfere with tumor promoter-induced differentiation. Our findings also sustain previous suggestions that the oncogenes v-myb and v-myc may not transform target cells by simply blocking differentiation.

Avian oncovirus Mill Hill No. 2: pathogenicity in chickens.

Mill Hill No. 2 (MH2), an avian tumor virus, was studied for its transforming and oncogenic effects. In tissue culture it induced transformation of chicken fibroblasts and yolk sac macrophages. When injected into the chicken, the main feature of this virus was its ability to cause liver and kidney carcinoma, in addition to sarcoma. MH2-associated viruses did not transform cell cultures but were able to cause only lymphoma in the birds. Light and electron microscopy were used in a detailed histologic study of the tumors induced by MH2 virus. An unclassified round cell sarcoma was produced in soft tissues at sites of injection; there was no evidence of origin from endothelium. In the kidney, carcinomas mixed with a malignant stroma were found. Hepatocarcinomas were the dominant tumors found in the liver. The lymphomas produced by the associated virus were poorly differentiated and highly malignant. The study illustrated the highly oncogenic potential of this virus and offered a model for the analysis of the carcinogenic events in a more specific way.

BM2L is a spontaneous leukemogenic variant of a non-leukemogenic v-myb-transformed myeloid cell line.

Leukemogenesis is a complex process involving an accumulation of genetic lesions affecting both growth and differentiation in cells of the hematopoietic lineage. Our laboratory has established a non-producer v-myb-transformed cell line (BM2/C3A) which, when injected into the chicken embryo, does not produce leukemia. Recently, a spontaneous variant of this cell line, called BM2L, was obtained from in vivo experiments. BM2L produces an acute monoblastic leukemia when injected into the chicken embryo. BM2L cells do not differentiate in vivo or in vitro, but continue to proliferate under conditions in culture that allow for the differentiation of BM2/C3A cells into macrophages. In addition, BM2L cells have reduced requirements for exogenous growth factors. BM2L cells contain the v-myb allele and express v-Myb protein, but leukemogenicity does not involve point mutations in v-myb. The BM2 model, consisting of two non-producer cell lines differing in vivo in their leukemogenicity, provides a novel system for identifying genes that play a role in the induction or suppression of leukemogenesis.

Leukemogenicity of v-myb-transformed monoblasts cells can be modulated by normal bone marrow environment.

The avian myeloblastosis virus (AMV) causes monoblastic leukemia in the chick. Two non-producer clones of AMV-transformed monoblasts, BM2/C3A and BM2L/A2B5, have been described (see Bottazzi et al., this issue). They differ in their growth requirements and in their ability to induce leukemia when injected into the chick embryo. We first genetically tagged these clones by retroviral infection with a vector expressing the bacterial lacZ gene. Then, we injected the lacZ-positive cells via the chorioallantoic vein into chick embryos. With BM2L/A2B5 cells, the bone marrow of the injected birds was rapidly invaded by lacZ-positive cells. In addition, these cells rapidly overgrew cultures of bone marrow cells derived from injected animals. Conversely, the growth of BM2/C3A was inhibited in the injected animals and only a few blue cells, with the morphology of macrophages, were detected in cultures of bone marrow cells. We developed an in vitro assay to mimic in vitro the differential growth of BM2/C3A and BM2L/A2B5 observed in vivo. These data strongly suggest that BM2/C3A cells retain their ability to differentiate into macrophages in the normal bone marrow environment and that BM2L/A2B5 cells differ from BMC/C3A in the loss of this capacity.

Genetic variation and host markers in the src gene of recovered avian sarcoma viruses.

The src genes of three recovered avian sarcoma viruses were compared by RNase T1 oligonucleotide fingerprinting and tryptic peptide analysis. In all three recovered avian sarcoma viruses the oligonucleotide composition of src was different and also distinct from that of the parental Schmidt-Ruppin strain of Rous sarcoma virus. This evidence for genetic variation src was strengthened by two dimensional peptide maps of the src gene products pp60src, translated in a reticulocyte lysate system in vitro. Numerous differences between the peptide patterns of the pp60src proteins produced by the parental and the recovered viruses were detected. No two src proteins were identical, while the tryptic peptide maps of the internal gag proteins synthesized by these viruses were indistinguishable. The src proteins of recovered avian sarcoma viruses also contained peptides that were absent from the src protein of parental Schmidt-Ruppin D virus but were found in the endogenous src protein of normal cells. We conclude that there is considerable genetic variation in the src gene of recovered avian sarcoma viruses and that these recovered src genes contain host cell-derived markers.

Effect of avian myeloblastosis virus in the Japanese quail.

Moscovici, Carlo (University of Colorado Medical Center, Denver), and E. H. Macintyre. Effect of avian myeloblastosis virus in the Japanese quail. J. Bacteriol. 92:1141-1149. 1966.-Avian myeloblastosis virus (AMV) induced a spectrum of neoplasms in Japanese quail (Coturnix coturnix japonica) which was similar to that observed in the chicken, with one exception: the total absence of acute myeloblastic leukemia in quail. Studies in vivo as well as in vitro suggested that the cause for this difference may be ascribed to the heterogeneity of AMV and to the genetic makeup of the quail cell.

Isolation and characterization of a temperature-sensitive mutant of avian myeloblastosis virus.

A temperature-sensitive (ts) mutant, GA 907/7, was isolated after mutagen treatment of avian myeloblastosis virus. When bone marrow cells or secondary yolk sac macrophages were infected with GA 907/7, the expression of transformation was greatly reduced at 41 degrees C. The results of temperature-shift experiments suggest that in GA 907/7 the putative v-myb gene product is functional only at 35.5 degrees C. Moreover, when ts-induced transformed cells were shifted to 41 degrees C, a partial morphological conversion to macrophage-like cells was obtained, while the majority of the cells underwent senescence and lysis. No leukemia was obtained when GA 907/7 was injected in 1-day-old chickens. Finally, a continuous cell line releasing genetically stable mutant virus was obtained after transformation of secondary yolk sac cells.

Genetic structure of avian myeloblastosis virus, released from transformed myeloblasts as a defective virus particle.

Chicken myeloblasts transformed by avian myeloblastosis virus (AMV) in the absence of nondefective helper virus (termed nonproducer cells) were found to release a defective virus particle (DVP) that contains avian tumor viral gag proteins but lacks envelope glycoprotein and a DNA polymerase. Nonproducer cells contain a Pr76 gag precursor protein and also a protein that is indistinguishable from the Pr180 gag-pol protein of nondefective viruses. The RNA of the DVP is 7.5 kilobases (kb) long and is 0.7 kb shorter than the 8.2-kb RNAs of the helper viruses of AMV, MAV-1 and MAV-2. Comparisons based on RNA.cDNA hybridization and mapping of RNase T1-resistant oligonucleotides indicated that DVP RNA shares with MAV RNAs nearly isogenic 5'-terminal gag and pol-related sequences of 5.3 kb and a 3'-terminal c-region of 0.7 kb that is different from that found in other avian tumor viruses. Adjacent to the c-region, DVP RNA contains a contiguous specific sequence of 1.5 kb defined by 14 specific oligonucleotides. Except for two of these oligonucleotides that map at its 5' end, this sequence is unrelated to any sequences of nondefective avian tumor viruses of four different envelope subgroups as well as to the specific sequences of fibroblast-transforming avian acute leukemia and sarcoma viruses of four different RNA subgroups. The specific sequence of the DVP RNA is present in infectious stocks of AMV from this and other laboratories in an AMV-transformed myeloblast line from another laboratory, and it is about 70% related to nucleotide sequences of E26 virus, an independent isolate of an AMV-like virus. Preliminary experiments show DVP to be leukemogenic if fused into susceptible cells in the presence of helper virus. We conclude that DVP RNA is the leukemogenic component of infectious AMV and that its specific sequence, termed AMV, may carry genetic information for oncogenicity. Thus we have found here a transformation-specific RNA sequence, unrelated to helper virus, in a highly oncogenic virus that does not transform fibroblasts.

Molecular cloning of the avian erythroblastosis virus genome and recovery of oncogenic virus by transfection of chicken cells.

Avian erythroblastosis virus (AEV) causes erythroblastosis and sarcomas in birds and transforms both erythroblasts and fibroblasts to neoplastic phenotypes in culture. The viral genetic locus required for oncogenesis by AEV is at present poorly defined; moreover, we know very little of the mechanism of tumorigenesis by the virus. To facilitate further analysis of these problems, we used molecular cloning to isolate the genome of AEV as recombinant DNA in a procaryotic vector. The identity of the isolated DNA was verified by mapping with restriction endonucleases and by tests for biological activity. The circular form of unintegrated AEV DNA was purified from synchronously infected quail cells and cloned into the EcoRI site of lambda gtWES x B. A restriction endonuclease cleavage map was established. By hybridization with complementary DNA probes representing specific parts of avian retrovirus genomes, the restriction map of the cloned AEV DNAs was correlated with a genetic map. These data show that nucleotide sequences unique to AEV comprise at least 50% of the genome and are located approximately in the middle of the AEV genome. Our data confirm and extend previous descriptions of the AEV genome obtained by other procedures. We studied in detail two recombinant clones containing AEV DNA: the topography of the viral DNA in the two clones was virtually identical, except that one clone apparently contained two copies of the terminal redundancy that occurs in linear viral DNA isolated from infected cells; the other clone probably contained only one copy of the redundant sequence. To recover infectious virus from the cloned DNA, we developed a procedure for transfection that compensated for the defectiveness of AEV in replication. We accomplished this by ligating cloned AEV DNA to the cloned DNA of a retrovirus (Rous-associated virus type 1) whose genome could complement the deficiencies of AEV. Ligation of the two viral DNAs was facilitated by using a neutral fragment of DNA as linker between otherwise noncompatible termini. Cloned AEV DNA gave rise to infectious AEV capable of transforming fibroblasts and bone marrow cells in culture and of inducing both sarcomas and erythroleukemia in chickens. We conclude that the cloned DNAs represent the authentic genome of AEV undisturbed by the cloning procedure. Molecular cloning offers a powerful approach to the identification and characterization of retrovirus genomes.

Identification of a provirally activated c-Ha-ras oncogene in an avian nephroblastoma via a novel procedure: cDNA cloning of a chimaeric viral-host transcript.

Retrovirus without oncogenes often exert their neoplastic potential as insertional mutagens of cellular proto-oncogenes. This may be associated with the production of chimaeric viral-host transcripts; in these cases; activated cellular genes can be identified by obtaining cDNA clones of bipartite RNAs. This approach was used in the analysis of chicken nephroblastomas induced by myeloblastosis-associated virus (MAV). One tumor contained a novel mRNA species initiated within a MAV LTR. cDNA cloning revealed that this mRNA encodes a protein of 189 amino acids, identical to that of normal human Ha-ras-1 at 185 positions, including positions implicated in oncogenic activation of ras proto-oncogenes; there are no differences between the coding sequences of presumably normal Ha-ras cDNA clones from chicken lymphoma RNA and the tumor-derived cDNAs. The chimaeric mRNA in the nephroblastoma is at least 25-fold more abundant than c-Ha-ras mRNA in normal kidney tissue, and a 21-kd ras-related protein is present in relatively large amounts in the tumor. We conclude that a quantitative change in c-Ha-ras gene expression results from an upstream insertion mutation and presumably contributes to tumorigenesis in this single case. Little or no increase in c-Ha-ras RNA or protein was observed in other nephroblastomas.

Embryonic erythroid cells transformed by avian erythroblastosis virus may proliferate and differentiate.

Embryonic chick cells from the primitive streak stage to later stages of the developing embryo were infected with avian erythroblastosis virus (AEV). The data indicate that the greatest number of target cells for AEV was observed in the 12-somite blastoderm and gradually decreased in hemopoietic tissues with the development of the embryo. The target cell for AEV is not in the BFU-E compartment, as it is in the adult bone marrow, but is probably recruited within the CFU-M compartment which precedes the BFU-E compartment. Our studies also show that a significant number of transformed colonies derived from embryonic hemopoietic tissues undergo hemoglobinization in contrast with what is observed in transformed colonies of bone marrow. A complete characterization of the embryonic and adult hemoglobin is at present under study.

The origin of chicken hematopoietic colonies as assayed in semisolid agar.

This investigation was undertaken to determine whether primitive stem cells and/or fully differentiated macrophages were the source of in vitro colonies derived from hematopoietic tissues. The chicken colony-forming cell (CFC) present in uncultured yolk sac was a nonadherent, presumably undifferentiated cell. The efficiency of colony formation in this case was approximately 0.08%. In contrast to uncultured yolk sac, the CFC present in one-week old yolk sac cultures was evidently a macrophage. Yolk sac cultures, which consisted of greater than 99% macrophages, produced colonies with an efficiency of 1-5% while cultures derived from peritoneal macrophages produced colonies with an efficiency of 10%. Silica selectively destroyed macrophages and reduced the colony forming efficiency of cells derived from yolk sac cultures.

Leukemogenicity of avian oncovirus S13.

Avian oncovirus S13 induces erythroblastic and granulocytic leukemias in line 6 and Spafas chickens. It also causes anemia, sarcomas, and endothelial proliferation. The leukemic cells contain the transformation-specific protein of S13, gp155.

Site-specific mutagenesis of avian erythroblastosis virus: v-erb-A is not required for transformation of fibroblasts.

Avian erythroblastosis virus (AEV) is an acutely transforming retrovirus whose putative oncogenes (v-erb-A and v-erb-B) encode the proteins P74gag-erb-A and P61-68erb-B. The existence of these two gene products has prompted the question of whether one or both proteins are required in the transformation of erythroblasts and fibroblasts by AEV. In the accompanying manuscript, we describe the use of site-specific mutagenesis to generate mutants of AEV unable to synthesize P61-68erb-B. Here we present our analysis of the oncogenic potential of an AEV mutant unable to synthesize P74gag-erb-A due to a large deletion encompassing both gag and v-erb-A sequences. The erb-A-mutant retrovirus propagated quite poorly on fibroblasts in culture; however, fibroblasts harboring the erb-A mutant genome were transformed in the absence of P74gag-erb-A expression. The mutant virus failed to induce erythroleukemias in chickens, but the validity of this finding is compromised by the poor replicative capacity of the mutant. The results presented in this and the preceding manuscript indicate that P61-68erb-B is both necessary and sufficient for neoplastic transformation of fibroblasts by AEV; by contrast, a role for p74gag-erb-A in leukemogenesis by AEV has not yet been rigorously excluded.

Response of hemopoietic cells to avian acute leukemia viruses: effects on the differentiation of the target cells.

Chicken bone marrow cells were infected with three avian acute leukemia viruses (ALV)--avian myeloblastosis virus (AMV), myelocytomatosis virus strain MC29 and Mill Hill 2 virus (MH2)--and then cultured in agar in the presence of conditioned medium. Under these conditions, it was found that very few cells served as target cells for these three viruses. Density gradient separation showed that ALV target cells were found primarily in the light density fractions and might be represented by cells committed to the mononuclear phagocyte pathway. Separation of bone marrow cells on the basis of their sedimentation velocity at unit gravity suggested that MC29 and AMV did not share the same target cells. In addition, the analysis of surface receptors and functional markers characteristic of macrophages (Fc and complement receptors, phagocytosis and immune phagocytosis) indicated that the ALV-transformed cells were blocked during their differentiation. These results indicate that the transforming ability of ALV interferes with the differentiation of their target cells.

Persistence of avian oncoviruses in chicken macrophages.

Inoculation of avian oncoviruses into 1- to 2-month old chickens led to a rapid production of antiviral humoral antibodies. Under these conditions it was found that avian leukosis viruses are sequestered in macrophages of peripheral blood, in which they can persist for a long period of time (up to about 3 years). In contrast, avian sarcoma viruses were never found in macrophages from chickens during the progression of sarcomas or after regression of the tumors.