relate with the biological properties of mutant and revertant viruses, we ... To study the biological activity of the v-erbA and v-erbB genes of td359 and r12, and to ...
The EMBO Journal vol.6 no.2 pp.375-382, 1987
A single point mutation in erbA restores the erythroid transforming potential of a mutant avian erythroblastosis virus (AEV) defective in both erbA and erbB oncogenes
Klaus Damm, Hartmut Beug, Thomas Graf and Bjorn Vennstrom Differentiation Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900 Heidelberg, FRG Communicated by T.Graf
We have characterized the v-erbA and v-erbB oncogenes of td359, a transformation-defective mutant of avian erythroblastosis virus (AEV) unable to transform erythroblasts, and the revertant r12, obtained after in vivo passage of the mutant. Molecular cloning, sequencing, construction of chimeric viruses and testing of their oncogenic capacities revealed that both oncogenes of td59 are mutated and biologically defective. The r12 virus, although still containing a mutant v-erbB gene, recovered its erythroid transforming potential by acquiring a highly active gag-erbA gene. These results demonstrate that two co-operating oncogenes, an active v-erbA and a defective v-erbB, can transform a cell type not transformed by either oncogene alone. Furthermore, a single amino acid substitution inactivated the td359 v-erbA protein and we show that its reversion led to the reactivation of the protein. This lesion is located in the same region as several previously described inactivating mutations of glucocorticoid receptors, suggesting that the structure/function relationship of the virally transduced form of the cerbA/thyroid hormone receptor is closely siumlar to that of steroid hormone receptors. Key words: erbA/erbB/oncogenes/avian erythroblastosis virus /single point mutation Introduction Several examples of co-operativity between oncogenes in cell transformation have been described previously (for review, see Weinberg, 1985). The avian erythroblastosis virus AEV-ES4 provides another example of oncogene co-operativity: both of its oncogenes, v-erbA and v-erbB, contribute to the transformed phenotype of infected erythroid cells. The v-erbA oncogene has no detectable transforming activity on its own, while v-erbB is sufficient for transformation of both erythroblasts and fibroblasts in vivo and in vitro (Frykberg et al., 1983; Sealy et al., 1983). However, erythroblasts transformed by v-erbB alone differentiate spontaneously and require specific culture conditions for propagation in vitro, whereas erythroblasts expressing v-erbA in addition to v-erbB are completely arrested at an early stage of differentiation and grow in standard tissue culture media (Frykberg et al., 1983; Beug et al., 1985; Kahn et al., 1986). Recently we found that erythroblasts induced to self-renew by infection with viruses containing v-src, v-sea, v-fps and v-rasH oncogenes are likewise blocked in their spontaneous differentiation upon superinfection with v-erbA. This suggests that v-erbA co-operates with these diverse types of oncogenes in a manner very similar to that seen with v-erbB, and that the two types of oncogenes co-operate by influencing independent physiological ( IRL Press Limited, Oxford, England
pathways (Kahn et al., 1986). This notion is further supported by the recent identification of the cellular homologs to the oncogenes of AEV: c-erbA encodes a high affinity nuclear receptor for thyroid hormone (Sap et al., 1986), whereas c-erbB is the gene for the epidermal growth factor receptor (Downward et al., 1986; Ullrich et al., 1984; Weinberger et al., 1986). We have previously described a transformation-defective mutant of AEV, denoted td359, which has lost the capacity to cause leukemia and to transform erythroblasts, although it still transforms fibroblasts and induces sarcomas (Royer-Pokora et al., 1979; Beug et al., 1980). As shown here, however, in rare instances td359-infected chicks develop erythroleukemia, and from one such an animal we isolated a revertant virus (designated r12) that had regained erythroblast transforming capacity. To localize and characterize the lesions in td359 and in r12, we cloned and sequenced both viral genomes and constructed recombinant viruses with new oncogene combinations. Tests of their erythroid transforming potential revealed that both oncogenes are defective in td359, and that rl2 recovered its erythroid transforming potential due to the reversal of a single point mutation in v-erbA of td359. These findings indicate that v-erbA can restore the erythroid transforming capacity of a defective v-erbB gene. Results Isolation and characterization of a revertant virus after in vivo passage of td359 In an attempt to obtain revertants of td359, virus was injected intravenously into 12-day-old chick embryos. Of 25 chicks hatched, two developed an erythroblastosis -3.5 weeks after injection. From these two animals transformed erythroblast cultures could be established with properties similar to those transformed by AEV-ES4 (referred to as wtAEV in this paper) and producing viruses that had regained the capacity to transform erythroblasts in vitro and to induce erythroleukemias with incidences and latencies comparable to wtAEV (T.Graf, unpublished observations). One of these two virus isolates, named rl2, was chosen for further study. To obtain a first clue about the molecular changes that correlate with the biological properties of mutant and revertant viruses, we compared the gag-erbA and the v-erbB proteins from cells transformed by wtAEV, td359 or r12. The protein encoded by v-erbAwt is a gag -erbA fusion protein of 75 kd that can be cleaved by p15 retroviral protease into a gag fragment of 30 kd and a v-erbA fragment of 45 kd. The corresponding protein of td359 has a mol. wt of 74 kd and cleaves into a F3Ogag and a F44erbA fragment (Beug et al., 1980), whereas the v-erbA fragment of r12 had regained a size similar to that obtained from v-erbAwt (Figure 1). The v-erbBwt protein is synthesized as an unglycosylated precursor of 62 kd and processed to 68- and 74-kd forms after glycosylation (Hayman et al., 1983; Hayman and Beug, 1984). As shown in Figure 1, the td359 and rl2 viruses expressed verbB proteins that are 12 kd smaller than the v-erbBwt pro375 -
K.Damm et al.
erbA wt
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r12
wt
td
r12 -99
)
69
gP68{
-- 69
1
F45 -. X FA 5 --/
55-45
gp 56{ 'II
-45 F30 -2C)(:
Fig. 1. Analysis of mutant and revertant v-erbA and v-erbB proteins. [35S]Methionine labeled extracts of wtAEV, td359 and rl2 transformed cells were immunoprecipitated with v-erbA- and v-erbB-specific antiserum. The immunoprecipitated gag-erbA proteins were then cleaved with p15 protease to distinguish the gag and erbA domains. Values on the right-hand side mark the position of 14C-labeled mol. wt standards.
tein. As revealed by immunofluorescence, the cell surface expression of mutant and revertant v-erbB proteins were indistinguishable from that of the v-erbBwt protein (data not shown). These results suggest that td359 had suffered at least two alterations, a large truncation in the v-erbB gene in addition to a minor change in the v-erbA fragment of p75 described earlier (Beug et al., 1980). The v-erbA protein of the revertant r12 had recovered an electrophoretic mobility similar to that of wtAEV. To allow a determination of the nature of the mutations in td359 and the backmutation(s) in r12, both viral genomes were molecularly cloned. The correct arrangement of the mutant viral genomes was verified by restriction endonuclease mapping, hybridization analysis and nucleotide sequencing (shown below). Figure 2 demonstrates that the genome size and restriction map of both td359 and r12 differ from that of wtAEV (clone AEV-1 1, Vennstrom et al., 1980). Most strikingly, they exhibit a significant deletion in the v-erbB domain, an observation which supports the protein data. The fact that both td359 and r12 have EcoRI sites in their LTRs is likely to be due to recombination with the RAV-1 helper virus [which has an EcoRI site in its U3 sequence, Payne et al. (1981)] used for the propagation of these viruses (Royer-Pokora et al., 1979). In contrast, AEV-1 1 was cloned from a stock with a helper virus lacking EcoRI sites in its U3 sequence (Vennstrom et al., 1980). Erythroid transforming capacities of chimeric viruses containing different combinations of v-erbA and v-erbB of td359, r12 and wtAEV To study the biological activity of the v-erbA and v-erbB genes of td359 and r12, and to determine how the respective mutations in these oncogenes contribute to the observed mutant and revertant phenotypes, recombinant viruses were constructed containing all nine possible combinations of the wtAEV, td359 and r12 v-erbA and v-erbB oncogenes. The details of the constructions are described in Materials and methods. A schematic illustration of the resulting recombinant genomes is shown in Figure 3. The biological activity of the chimeric genomes was 376
XhoI wtAEV
1u3N:::>A gag
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PvuII SaIlI ApaI
, K l.l :>. A ga'g :. ' 'e'rbA
B
AA env
U3 RI
AerX b''B' A env lU3
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Fig. 2. Restriction maps of cloned mutant and revertant AEV genomes. The figure shows only a small selection of sites including those used for cloning and identification of the mutant genomes.
determined by transfection of chicken embryo fibroblasts with the plasmids described above together with cloned RAV-1 helper virus DNA (Frykberg et al., in preparation). All virus constructs transformed fibroblasts and gave rise to foci, some of which were isolated and expanded in liquid culture. The erythroid transforming capacities of the virus constructs were determined by cocultivating the virus-producing, transformed fibroblast cultures with chick bone marrow cells in CFU-E medium. This medium promotes growth of erythroblasts transformed by v-erbB in the absence of v-erbA (Frykberg et al., 1983; Beug et al., 1985). Seven of the nine constructs were able to generate proliferating mass-cultures of transformed erythroblasts (Figure 3). In addition, culture supernatants were capable of inducing the formation of 50-200 transformed colonies per ml after infection of bone marrow cells and seeding them in Methocel (data not shown). The remaining two constructs, both containing the v-erbA gene from td359 and the v-erbB gene derived from either td359 or r12, failed to transform erythroblasts. These viral constructs induced the transient appearance of immature hematopoeitic cells which, although expressing the v-erbBtd protein, differentiated terminally within 10-12 days after co-cultivation.
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Origin of viral oncogenes
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Fig. 3. Erythroid transforming activities of recombinant viruses carrying wild-type, mutant and revertant v-erbA and v-erbB oncogenes. Plasmids containing all nine possible combinations of the wtAEV, td359 and r12 verbA and v-erbB genes are shown. The XhzoI site used to construct the chimeric viruses is located within the gag-domain, the ApaI site is located at the v-erbB splice acceptor site and the EcoRI site is located in the 3' LTR. Open boxes mark genomic fragments derived from wtAEV, black boxes mark genomic fragments derived from td359 and dotted boxes mark genomic fragments derived from r12. + stands for generation of transformed colonies >50 CFU/ml, stands for inability to generate continuously proliferating cultures of erythroblasts. -
Immunoprecipitations of erythroblasts transformed by the various constructs showed that they expressed v-erbB proteins of the expected sizes. However, some constructs showed a slight mol. wt increase in the gag fragments of their gag-erbA protein, suggesting recombination events with the gag gene of the helper virus. Similar alterations in the gag region of the v-erbA fusion protein have frequently been observed with various cloned AEV-viruses (H.Beug and B.Vennstrom, unpublished observations). In summary, these data show that viruses with the v-erbAtd gene in combination with the v-erbBtd or v-erbBr12 genes are defective for erythroblast transformation, whereas viruses with either v-erbAwt or v-erbAr12 in combination with any of the three v-erbB genes are capable of transforming erythroblasts. These results indicate that both v-erbAwt and verbAr12 can restore the erythroid transforming capacity of a defective v-erbB gene. Differentiation phenotypes of erythroblasts transformed by the recombinant viruses To characterize the biological properties of the transformation competent viral constructs in more detail, transformed erythroblasts were analysed for their capacity to undergo spontaneous maturation to erythrocytes. For this purpose a number of independently transformed erythroblast clones were isolated from methocel cultures and tested for the proportion of hemoglobin-positive cells using a highly sensitive benzidine stain-
s.
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Fig. 4. Spontaneous differentiation of erythroblasts transformed by the recombinant viral genomes. Twenty-four individual transformed colonies of erythroblasts each (nine for erbA-erbB't) were expanded in CFU-E medium and analyzed for hemoglobin expression by staining with acid benzidine. Individual clones were arbitrarily plotted in decreasing order according to their proportion of hemoglobin positive cells. Nomenclature of constructs has been abbreviated such that the designation 'erb' was omiitted.
ing method. The data, shown in Figure 4, allow the following conclusions to be drawn: (i) erythroblasts transformed by the erbAtderbBwt virus resemble cells transformed by AEV-H, a virus containing v-erbB only (labeled A-Bwt in the Figure), in that they exhibited a high proportion of spontaneously differentiating cells: in 11 of 24 clones analyzed, > 20% of the cells were positive for hemoglobin. This indicates that the v-erbAtd protein is biologically inactive or grossly impaired in its function. (ii) erythroblast clones transformed by viral constructs containing the v-erbAwt gene in combination with either v-erbBtd or v-erbBrl2 showed a higher proportion of hemoglobin-positive cells (in each case seven out of 24 clones contained > 20% positive cells) than those transformed in combination with verbBwt. This indicates that the v-erbAwt gene partially restores the erythroblast transforming ability of the defective v-erbB genes of td359 and r12 and that the latter are impaired but not inactive in their ability to induce erythroblast proliferation. (iii) erythroblast clones transformed by viral constructs containg the v-erbArl2 gene in combination with any of the three verbB genes showed virtually no spontaneous differentiation, as 377
K.Danm et al.
Table I. Expression of erythroid differentiation markers in erythroblasts transformed by chimeric viral constructs
Virus construct
number of clones containing
Virus construct
late reticulocytes/ mature erythrocytesb
erythrocyte antigen positive cells
Table II. Induction of acute erythroblastosis by viral constructs containing the v-erbA gene of wtAEV and r12 in combination with v-erbBtd erbA
erbB
wt
td td
r12
erbA
erbB
5-20%a
>20%
wt wt wt td rl2 rl2 rl2
wt td rl2 wt wt td r12
2/8 4/8 2/6 3/8 1/8 2/8 ND
0 1/8 4/6 1/8 0 0 ND
+ + +
-
wtC
0
5/5
+
Incidence of erythroblastosisa
Latency period ofb erythroblastosis eyholsoi
death et
10/15 10/14
17 (13-23) 16 (13-20)
28 (16-36) 24 (20-27)
aNumber of positive animals per total number of animals tested bLatency in days (range) 1623
317
V
...GATGAGTATCTTGTC ... AspGluTyrLeuVal
1317
anumber of clones containing the indicated percentages of positive cells per total number of clones analyzed. b_ stands for
