from those for MC29 (6) and that the former are more immature than the latter. The transformation target cell specificity of DLVs appears

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 77, No. 1, pp , January 1980 Cell Biology Target cell specificity of defective avian leukemia viruses: Hematopoietic target cells for a given virus type can be infected but not transformed by strains of a different type (acute defective leukemia viruses/cell transformation/gag-related proteins) THOMAS GRAF*, HARTMUT BEUG*, AND MICHAEL J. HAYMANt *Institut fur Virusforschung, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 6900 Heidelberg, West Germany; and timperial Cancer Research Fund Laboratory, Lincoln's Inn Fields, P.O. Box 123, London WC2A 3PX, England Communicated by Wolfgang Beermann, September 18, 1979 ABSTRACT Defective avian leukemia viruses of the avian erythroblastosis (AEV), avian myelocytomatosis (MC29), and avian myeloblastosis (AMV) type induce the proliferation of leukemic cells with properties of erythroblasts, macrophages, and myeloblasts, respectively. Their target cells can be separated and have properties of cells of the erythroid (AEV) and myeloid lineage (MC29 and AMV), respectively. In the present study we have shown that this target cell specificity is not due to the ability of the different strains to infect only certain types of hematopoietic cells. Instead, AEV was found to replicate in macrophages and to induce the expression of p75 AEV, its presumptive transforming protein. Likewise, MC29 was found to replicate in AEV-infected erythroblasts as well as in AMV-infected myeloblasts and to express the p110 MC29 protein in these cells. Superinfection with MC29 or AMV of ts34 AEVinfected erythroblasts did not impair their capacity to accumulate hemoglobin sfter shift to nonpermissive temperature. Our results support a model in which the transforming proteins of AEV, MC29, and AMV block the differentiation of their target cells by competitively inhibiting the action of a hypothetical homologous cellular differentiation protein synthesized in the corresponding target cells only. Replication-defective avian leukemia viruses (DLVs) are a group of oncoviruses isolated from the domestic chicken that are capable of causing acute leukemias and other types of neoplasms within weeks or months after infection. A unique property of these viruses is their specificity of transformation in the hematopoietic system in vivo and in vitro (1). Accordingly, they have been subdivided into avian erythroblastosis virus (AEV)-type strains, avian myelocytomatosis virus (MC29)-type strains, and avian myeloblastosis virus (AMV)-type strains (1). Thus, AEV causes erythroblastosis in vivo and induces the proliferation of erythroblast-like cells after infection of bone marrow cells in vitro (1-3). The four MC29-type strains (MC29, CMII, OK10, and MH2) induce myelocytomatosis or related myeloid leukemias [with the possible exception of OK10 (1)] and induce the proliferation of macrophage-like transformed bone marrow cells in vitro. AMV and E26, the two AMV-type strains, induce myeloblastosis in vivo and myeloblast-like transformed cells in vitro (1, 3). The apparent cause for this lineage specificity of DLVs is the observation that they selectively transform certain types of normal target cells in the bone marrow. Thus, the target cells for transformation by AEV on the one hand and by MC29 and AMV on the other can be separated before infection and express markers of the erythroid and myeloid lineage of differentiation, respectively (refs. 4 and 5; unpublished data). In addition, recent evidence suggests that the target cells for AMV are different from those for MC29 (6) and that the former are more immature than the latter. The transformation target cell specificity of DLVs appears to be independent of the helper virus used (1). In the case of the myeloid strains, however, helper viruses of subgroups B or C only have been reported to confer on them the capacity of infecting and transforming hematopoietic cells (7). What is the mechanism of target cell specificity of these viruses in the hematopoietic system? One obvious possibility is that AEV can infect and replicate only in erythroid cells, whereas MC29- and AMV-type viruses cap do so only in myeloid cells at apparently different stages of differentiation. To test this, ideally, all three types of target cells should be infected with AEV, MC29, and AMV, the prototype DLV strain of each group, and then assayed for virus expression. Unfortunately, it seems unlikely that pure populations of target cells can be obtained in the near future because AEV, MC29, and AMV transform only about 100, 2500, and 500 target cells out of 106 chick bone marrow cells, respectively (unpublished observations and ref. 5). To circumvent this problem we have used two alternative approaches. First, we tested the replication capacity of AEV in macrophages, the only type of normal avian hematopoietic cell that can be maintained in culture. These cells, although being totally refractory to transformation by AEV, still contain a high number of target cells for MC29 and, depending on the preparation used, also for AMV (4-6). A unique feature of the avian leukemia virus system is that, as mentioned above, hematopoietic target cells of different lineages can be transformed and cultured in vitro. The second approach, therefore, consisted in using nonproducer erythroblasts transformed by AEV and nonproducer myeloblasts transformed by AMV for infection studies with heterologous DLVs. This strategy was justified by the assumption that hematopoietic cells transformed by DLVs are very similar to their uninfected target cells, the main difference being that they are blocked in differentiation (1, 8, 9) and that they are able to proliferate. In this paper we demonstrate that DLVs replicate in nontarget cells and that their presumptive transforming proteins are expressed in these cells. Furthermore, results are presented that suggest that, in contrast to AEV, myeloid leukemia viruses do not interfere with the differentiation capacity of erythroblasts transformed with a temperature-sensitive mutant of AEV. Abbreviations: DLV, defective leukemia virus; AEV, avian erythroblastosis virus; ts34 AEV, a temperature-sensitive mutant of AEV; MC29, avian myelocytomatosis virus 29; AMV, avian myeloblastosis The publication costs of this article were defrayed in part by page virus; RAV and MAV, nondefective (helper) avian leukemia viruses; charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate focus-forming units; PFU, plaque-forming units; CFU, colony-forming RSV, Rous sarcoma virus; CEF, chicken embryo fibroblasts; FFU, this fact. units. 389

2 390 Cell Biology: Graf et al. MATERIALS AND METHODS Viruses and Cells. The origins of AEV(RAV-2), ts34 AEV- (RAV-2), MC29(RAV-2), and RAV-2 (Rous-associated virus, a helper avian leukemia virus) have been described (2, 9-11). These viruses were harvested from transformed bone marrow cells and Millipore filtered to remove cells before use. AMV- (MAV) of the BAI/A strain (10) was obtained from the plasma of moribund chicks infected with the virus. It contains a mixture of helper viruse's (MAV) of subgroups A and B. Cultures of normal macrophages and of transformed nonproducer clones derived from bone marrow infected with AEV, ts34 AEV, AMV, or MC29 were obtained as described (9, 10, 12). Cells were grown in Dulbecco's modified Eagle's medium containing 8% fetal calf serum, 2% chicken serum, 10 mm Hepes buffer at ph 7.3, and, in the case of AEV erythroblasts, with 10 AM thioglycerol added. Virus Infectivity Assays. The focus-plaque assay using chicken embryo fibroblasts (CEF) was performed as described (11). It was used to determine the fibroblast-transforming activity of AEV and MC29 [expressed as focus-forming units (FFU)/ml] and the plaque-forming activity of the helper virus RAV-2 [expressed as plaque-forming units (PFU)/ml]. The bone marrow transformation assay in semisolid. medium was performed essentially as described (5). Briefly, 10 X 106 bone marrow cells from 1- to 3-week-old chicks were suspended in 0.5 ml of growth medium and infected with 0.5 ml of virus. Then, 2.5 ml of growth medium containing 1.5% methylcellulose (Methocel) were added, and the cells were suspended and seeded in 35-mm dishes. This test was used to determine the hematopoietic transforming activity of AEV, MC29, and AMV. The titers obtained for AEV and MC29 were about 1/10 to 1/30 times as high in this assay as in the focus assay. Detection of gag-related Proteins in DLV-Infected Cells. Cells (5-10 X 106) were labeled with 200,ACi/ml of [ass]methionine (900 Ci/mmol, Amersham; 1 Ci = 3.7 X 1010 becquerels) for 1-2 hr at 37 C, and cell extracts were subjected to radioimmunoprecipitation analysis as described (13). A rabbit serum prepared against the Prague B strain of Rous sarcoma virus (PR-RSV-B) that reacts with the gag proteins p15, p19, and p27 as well as with the envelope antigen gp85 was used (13). Cytological Staining for Hemoglobin. The technique described by Orkin et al. (14) was employed. For each value, between 150 and 500 cells were scored. The standard deviation of five independent determinations of a sample of ts34 AEV erythroblasts containing an average of 86% benzidine-positive cells was +5.1%. RESULTS Replication of AEV in Macrophages. In a first set of experiments the replicating ability of AEV(RAV-2) in macrophages derived from chick bone marrow was determined. The cultures used were free of contaminating fibroblasts as determined by visual inspection and as judged by the fact that no colonies with fibroblast-like morphology developed within a period of 2 weeks. The AEV-infected macrophages were morphologically indistinguishable from those in an uninfected control culture and did not exhibit a significant increase in their rate of 2-deoxyglucose uptake as compared to the conttol. In another parallel culture infected with MC29(RAV-2), most of the initially adherent macrophages transformed into nonadherent, rapidly dividing cells by 1 week after infection. As can be seen from the results plotted in Fig. 1A, both AEV and its helper virus replicated in these cells at comparable rates, the helper virus always being at an approximately 10-fold excess..g 103 a..._ 0"10 c c Proc. Natl. Acad. Sci. USA 77 (1980).. I /..i.... I. L Time after infection, days FIG. 1. Replication kinetics of DLVs in nontarget cells. (A) Replication of AEV in a macrophage culture. Tertiary macrophages seeded at 5 X 105 cells in a 50-mm dish were infected with 0.5 ml of AEV(RAV-2) containing 2.5 X 105 FFU of AEV and 2 X 106 PFU of RAV-2. Cell supernatants were then harvested at daily intervals and assayed in the focus-plaque assay. 0, FFU/ml; 0, PFU/ml. (B) Replication of MC29 in AEV erythroblasts. Nonproducer erythroblasts transformed by ts34 AEV at 35 C (clone 24B1) were seeded at 1 X 106 cells in 35-mm dishes and superinfected with 0.2 ml of MC29(RAV-2) containing 2.4 X 105 FFU. The infected cells were washed once with growth medium 4 hr later and the supernatants were harvested at daily intervals thereafter. Samples were assayed for transforming activity in the colony-methocel assay with bone marrow cells. *, MC29-transformed colonies at 40 C; o, MC29- transformed colonies at 35 C; 0, ts34 AEV-transformed colonies at 35 C. Titers given are in colony-forming units (CFU)/ml. (C) Replication of MC29 in AMV myeloblasts. Nonproducer AMV myeloblasts (clone 33) grown at 37 C were seeded and superinfected with MC29 as described in the preceding section for ts34 AEV erythroblasts. Samples were assayed in the colony-methocel bone marrow transformation assay at 37 C. O, MC29-transformed colonies;,, AMVtransformed colonies. Titers given are in CFU/ml. Similar results were also obtained with a second AMV clone. The AEV infection experiment was repeated twice by infecting macrophage cultures obtained either from chick bone marrow or from peripheral blood of an adult chicken. Both experiments confirmed the results described above. Replication of MC29 and AMV in Transformed Nonproducer Erythroblasts and Myeloblasts. To determine whether erythroblasts are susceptible to infection with MC29 and AMV, nonproducer erythroblasts transformed with AEV in the absence of helper virus were used. If these cells are indeed susceptible they should produce not only the superinfecting myeloid virus strain and its corresponding helper virus but also AEV rescued by the helper virus. To distinguish between the two types of transforming viruses, nonproducer erythroblasts were used that had been transformed with ts34 AEV, a temperature-sensitive mutant of AEV. The samples obtained after superinfection of these cells were then assayed at 40 C to reveal colonies of the superinfecting DLV only, colony formation of ts34 AEV being suppressed at this temperature (9). In case of superinfection with MC29 the assay was also performed at 350C because colonies transformed by MC29 can easily be distinguished from ts34 AEV-transformed colonies (Fig. 2A). The results obtained after superinfection with MC29(RAV-2) of a clone of ts34 AEV erythroblasts are shown in Fig. 1B. As can be seen, MC29 seems to replicate to even higher titers than ts34 AEV produced in the same cells. In this experiment, the replication kinetics of the helper virus was not determined. That

3 i..4-7 Cell Biology:.. t.-, Graf et al. FIG. 2. Morphological distinction of colonies in semisolid medium transformed by different DLV strains. (A) Compact small (AEV) and diffuse large (MC29) colonies induced by progeny virus of MC29- superinfected ts34 AEV erythroblasts. Culture was incubated for 12 days at 350C. (B) Compact small (AMV) and large diffuse (MC29) colonies from progeny of MC29-superinfected AMV myeloblasts. Culture was grown at 37 C for 8 days. The nature of each colony type was verified by isolating several of them and by analyzing these clones for their expression of differentiation parameters as described (3). Bar represents 100,m. MC29 does not interfere with the replication of ts34 AEV is indicated by the finding that ts34 AEV was produced with an almost identical growth rate after superinfection of ts34 AEV erythroblasts with RAV-2 only (data not shown). Similar results were also obtained after superinfection with MC29 of another ts34 AEV- and an AEV-transformed nonproducer erythroblast clone. In the latter case MC29 synthesized was selectively assayed on macrophage cultures as described (12). The results obtained after superinfecting ts34 AEV erythroblasts with AMV(MAV) were less dramatic. Although a 7-fold increase was observed in the number of AMV colonies from the 1st to the 5th day of harvest, the maximum titer was only 4.4 X 101 CFU/ml (assay was at 40 C). The low titers obtained with AMV may reflect its property of replicating only poorly under in vitro conditions even when grown in myeloblasts (unpublished observations). In another set of experiments, nonproducer myeloblasts transformed by AMV were assayed for their capacity to support the growth of MC29(RAV-2). The results obtained are plotted in Fig. 1C. As can be seen, MC29 is able to replicate in myeloblasts although at a lower rate than in erythroblasts (Fig. 1B). AMV was produced at still lower titers, resembling results obtained after superinfection with helper virus only. Again, the replication kinetics of the helper virus itself was not determined. The myeloblasts superinfected with MC29 did not undergo any morphological changes towards that of MC29-transformed hematopoietic cells nor were they induced to express macrophage-specific antigen (3) on their surface. Synthesis of gag-related Proteins in DLV-Infected Nontarget Cells. Recently, Stehelin and coworkers (15, 16) have prepared cdna probes specific for AEV, MC29, and AMV. These probes hybridize with the RNA of all viruses of the corresponding type (e.g., the MC29 probe reacts with CMII, OK1O, and MH2) but not with RNA of viruses of the heterologous types or with the src gene of RSV. In addition, the AEV-, MC29-, and AMV-specific sequences are present in the DNA of normal cells (15-17). The strict correlation of these sequences to the transforming specificity of AEV-, MC29-, and AMV-type viruses led us to postulate that DLVs contain three new types of transforming genes, termed erb (for erythroblast), mac (for Proc. Natl. Acad. Sci. USA 77 (1980) 391 pro 8O- pr95-- pr76-._ p75 A macrophage), and myb (for myeloblast) (3, 8, 15, 16). Studies with ts34 AEV strongly suggest that, as with RSV, a viral gene product (probably a protein) is required for the maintenance of transformation by DLVs (5, 8, 9). Although the transforming proteins of DLVs have not yet been unequivocally identified, evidence has accumulated that a class of viral products referred to as gag-related proteins are responsible for transformation induced by avian (for review, see ref. 8) and mammalian DLVs such as the Abelson virus (18). These proteins, synthesized in all DLV-transformed cells, consist of part or all of the gag gene product and a unique portion. They exhibit sequence homologies correlating with the transforming specificity as well as with the specific RNA sequences of the different DLV strains (3, 8, 15, 26). In addition, MC29-specific RNA sequences have been shown to map at the position in the viral genome that corresponds to the location of the sequences coding for the gag-related protein of MC29 (19, 20). It is therefore likely that they represent the joint products of the gag gene and the viral oncogene. Thus, the 75,000-dalton protein induced by AEV (13), p75 AEV, presumably corresponds to a gag-erb protein, and the 110,000-dalton protein induced by MC29 (21), p110 MC29, to a gag-mac protein. The above-mentioned requirement of an AEV gene product for the maintenance of leukemic transformation rules out that a specific integration of DLVs within the genome of their target cells is sufficient to explain their target cell specificity. This raises the possibility that although DLVs can replicate in nontarget cells, their transforming proteins are not expressed in these cells and that this is the reason why no transformation ensues after infection. To determine whether or not p75 AEV and pl 10 MC29 are synthesized in infected nontarget cells, aliquots of cells used in experiments similar to those described in Fig. 1 were labeled and extracts were immunoprecipitated with an antiserum rea b c d e _~ WVb.4 '_ a b c d e f g h pr180 P_.0 - p _-- o pr76 m - p75 FIG. 3. Expression of gag-related proteins in nontarget cells infected with DLVs. (A) Expression of p75 AEV in macrophages. Lane a, extracts of AEV nonproducer fibroblasts [NP75 cells (13)] immunoprecipitated with rabbit anti-whole virus serum [VRS (13)]. Lanes b and c, extracts of AEV(RAV-2)-infected macrophages immunoprecipitated with VRS (b) and with normal rabbit serum (NRS) (c). Lanes d and e, uninfected macrophages immunoprecipitated with VRS (d) and with NRS (b). (B) Expression of p1 10 MC29 in erythroblasts. Lanes a and b, extracts of AEV nonproducer erythroblasts superinfected with MC29(RAV-2) and immunoprecipitated with VRS (a) and with NRS (b). Lanes c and d, AEV nonproducer erythroblasts superinfected with RAV-2 and immunoprecipitated with VRS (c) and with NRS (d). Lanes e and f, AEV nonproducer erythroblasts immunoprecipitated with VRS (e) and with NRS (f). Lanes g and h, MC29 nonproducer macrophage clone 14 immunoprecipitated with VRS (g) and with NRS (h). The latter two controls were run on a separate gel that included size markers. B

4 392 Cell Biology: Graf et al. Proc. Natl. Acad. Sci. USA 77 (1980) Table 1. Superinfection with MC29 and AMV does not interfere with capacity of ts34 AEV erythroblasts to differentiate Benzidine-positive cells, % 4 days 4 days 4 days Superinfecting Multiplicity at 350C, shifted to 41'C, shifted to 410C, virus of infection 4 days p.i. 4 days p.i. 8 days p.i. RAV-2 2.5* MC29(RAV-2) 0.25t AMV(MAV) 0.006t AEV(RAV-2) 0.Olt t p.i., Postinfection. * Number of PFU per cell. t Number of FFU per cell. Number of CFU per cell. Because the colony assay in bone marrow cells is more inefficient than the focus assay in fibroblasts, the effective multiplicity of infection with AMV was probably at least 10 times higher than the one indicated. acting with viral gag proteins. As shown in Fig. 3A, macrophages infected with AEV(RAV-2) express p75 AEV. In addition, they express the helper virus-induced gag-gene product pr76, the env gene product pr95, and the gag-pol gene product prl80. Similarly, wild-type AEV erythroblasts superinfected with MC29(RAV-2) expressed p110 MC29 in addition to p75 AEV, pr76, pr95, and prl80 proteins (Fig. 3B). In a repeat of the latter experiment using a clone of ts34 erythroblasts, a band in the 110,000-dalton region was observed also. AMV myeloblasts superinfected with MC29(RAV-2) yielded only a faint band in the 1 10,000-dalton region after immunoprecipitation (not shown), possibly reflecting the low replicating ability of MC29 in these cells. Superinfection of ts34 AEV Erythroblasts with MC29 and AMV Does Not Interfere with Their Capacity to Differentiate After Shift to Nonpermissive Temperature. Results descrijed earlier suggested that AEV blocks the differentiation of its hematopoietic target cells, because a shift from 35"C to 410C leads to an induction of hemoglobin synthesis in ts34 AEV-infected erythroblasts (9). We have postulated that this block is a consequence of the specific interaction of the transforming protein of AEV with differentiation factors of the target cell, and that this may be the basis for the target cell specificity for transformation by DLVs (1, 22). If this concept is correct, the transforming proteins of MC29 and AMV should have no effect on the capacity of erythroblasts to differentiate. Consequently, ts34 AEV-transformed erythroblasts superinfected with these viruses should still accumulate hemoglobin after shifting them from 35 C to 41 C, whereas superinfection with wild-type AEV should lead to another, superimposed, block of differentiation. To test this, ts34 AEV erythroblasts were superinfected with MC29(RAV-2) and AMV(MAV) and, as controls, with AEV- (RAV-2) and RAV-2, as described in Fig. 1 B and C and Fig. 2B. One set of cells was maintained at 350C, another was shifted for 4 days to 410C, and a third set of cells was first kept for 4 days at 350C and then shifted to 410C for 4 days. The latter schedule was chosen to give the superinfecting virus sufficient time to spread through the culture. The results obtained show that superinfection with MC29 or with AMV did not impair the differentiating ability of ts34 AEV erythroblasts, whereas superinfection with AEV interfered with the accumulation of hemoglobin (Table 1). A significant blocking effect of AEV was already observed when the virus was used at multiplicites of infection below the multiplicity of infection used for MC29 and AMV. That wild-type AEV replicates in ts34 AEV erythroblasts had been shown earlier (9). DISCUSSION The results presented show that AEV is capable of replicating in normal chick macrophages, that MC29 and AMV replicate in AEV erythroblasts, and that MC29 replicates in AMV myeloblasts. In addition, we found that the putative transforming proteins of AEV and MC29 are expressed in these hematopoietic nontarget cells. Because MC29 and AMV are capable of transforming only part of the macrophage population (5, 6), our experiments could be criticized by the assumption that AEV replicates only in that fraction of macrophages resistant to transformation by MC29 and AMV. Such an argument cannot be made for the results obtained from our second approach. That clones of erythroblasts transformed by AEV can be superinfected with both MC29 and AMV demonstrates that hematopoietic cells that are basically susceptible to leukemic transformation are also capable of expressing and replicating heterologous DLV strains. In these experiments, however, one could argue that DLVs are able to replicate in heterologous target cells only if these have been pretransformed. This possibility can be definitely ruled out only by infecting homogeneous populations of uninfected target cells with a heterologous DLV strain, a task that is hindered by the present lack of suitable methods for purification and maintenance in culture of normal avian hematopoietic cells other than macrophages. Nondefective leukemia virus of the RAV type cause predominantly lymphatic leukemia after a long period of latency. These viruses replicate in nontarget hematopoietic cells such as macrophages, erythroblasts, and myeloblasts, as has also indirectly been shown in this report. They are not comparable to DLVs, however, because they seem to lack an oncogene and probably transform hematopoietic cells by a different mechanism (23). Infection of macrophages with RSV leads to a replication of the virus and to slight changes in these cells (24). It also replicates in AEV erythroblasts with no apparent effects on their properties (ref. 9 and unpublished observations). It is not clear, however, whether or not the pp6o src protein of RSV is expressed in these cells. In the mammalian system, the defective Friend erythroleukemia virus can be grown in hematopoietic cells in the absence of an overt transformation (25). Again, the expression of a transforming protein in these cells remains to be shown. Defective avian leukemia viruses are capable of transforming not only certain types of hematopoietic cells but also cells from other tissues. AEV- and MC29-type viruses transform chicken fibroblasts whereas AMV does not (1, 10). In all cases the transformed cells support the growth of the infecting DLV. The replication ability of AMV in fibroblasts, however, has not yet been determined, due to the difficulty in obtaining clonepurified cultures of the corresponding cell types.

5 Cell Biology: Graf et al. AEV in target cell (erythroblast) Erythroblast Preceptor Cellular A ) -tiveblblock of erb protein L.A -...Jcomplex differentiation p75 AEV * *mi (gag-erb) 4 Transformation AEV in nontarget cell (macrophage) Macrophage receptor Cellular _ D- X fctive No effect on mac protein ; cvplex differentiation 01:D cd~~~~~~~~~~plex~~~~ p75 AEV P (gag-erb) A No transformation Fl(w. 4. Model explaining the lineage-specific transformation by I)LVs. In an infected erythroblast, p75 AEV (the presumptive gag-erb protein) competitively inhibits the reaction of a homologous cellular (erb) protein with its hypothetical erythroblast-specific receptor. This leads to the formation of an inactive p75 AEV-receptor complex and thus to a block of differentiation that in turn results in leukemic cell transformation. After infection with AEV of an immature macrophage (e.g., the target cell for MC29), p75 AEV finds no receptor and myeloid differentiation proceeds undisturbed. Conversely, in this model, infection of an immature macrophage with MC29 leads to a competitive inhibition by pl10 MC29 (the presumptive gag-mac protein) of the formation of a cellular mac protein-macrophage receptor complex. No effects are seen after infection of an erythroblast with MC29 due to the inability of p1 10 MC29 to find a receptor. The findings that DLVs are expressed in nontarget cells and that MC29 and AMV do not interfere with the differentiation capacity of target cells for AEV (in our case ts34 AEV erythroblasts) are compatible with the following hypothesis explaining the mechanism of transformation and hematopoietic target cell specificity of DLVs: The transforming proteins of DLVs block differentiation by competively inhibiting the action of a homologous cellular protein synthesized in the respective hematopoietic target cells only. A speculative diagram of the effects of AEV on a target and a nontarget cell in the light of this hypothesis is shown in Fig. 4. Our concept is based on recent findings that RNA sequences corresponding to the erb, mac, and myb genes of DLVs are present as single copies in the DNA of normal chicken cells and that they are conserved during evolution (16, 17). It is therefore likely that, as is the case for the src gene product (27, 28), normal cellular proteins will be found that are homologous to the proteins coded for by the oncogenes of DLVs. As discussed earlier, these proteins probably correspond to the unique portion of the gag-related proteins synthesized by DLVs. We have recently developed an antiserum specific for the non-gag (or erb) portion of p75 AEV (unpublished results); with this serum it may be possible to identify the hypothetical cellular erb protein in uninfected cells. If our model is correct we would also predict that this protein is erythroblast specific and that it is not expressed in nontarget cells such as in macrophages or myeloblasts. Proc. Nati. Acad. Sci. USA 77 (1980) 393 Note Added in Proof. Replication of MC29 virus and expression of p1 10 MC29 was also observed in ts34 AEV erythroblasts superinfected with MC29(RAV-2) and shifted to 400C for 4 days (unpublished data). We thank S. Grieser, G. Doederlein, and G. Kitchener for excellent technical assistance, and Dr. W. Meyer-Glauner for performing initial experiments on DLV replication in nontarget cells. 1. Graf, T. & Beug, H. (1978) Biochim. Biophys. Acta 516, Graf, T., Royer-Pokora, B., Schubert, G. E. & Beug, H. (1976) Virology 71, Beug, H., von Kirchbach, A., Doderlein, G., Conscience, J.-F. & Graf, T. (1979) Cell 18, Graf, T., Royer-Pokora, B. & Beug, H. (1976) in ICN-UCLA Symposium on Animal Virology, eds. Baltimore, D. & Huang, A. (Academic, New York), pp Graf, T., Beug, H., Royer-Pokora, B. & Meyer-Glauner, W. (1978) in Differentiation of Normal and Neoplastic Hematopoietic Cells, eds. Clarkson, B., Marks, P. A. & Till, J. E. 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