The ets Sequence Is Required for Induction of ... - Journal of Virology

Vol. 63, No. 1

JOURNAL OF VIROLOGY, Jan. 1989, p. 398-402

0022-538X/89/010398-05$02.00/0 Copyright © 1989, American Society for Microbiology

The ets Sequence Is Required for Induction of Erythroblastosis in Chickens by Avian Retrovirus E26 MICHAEL F. NUNNt AND TONY HUNTER* Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138-9216 Received 15 August 1988/Accepted 5 October 1988

E26 is a replication-defective avian retrovirus that causes an erythroblastic leukemia in vivo and transforms hematopoietic precursor ceils of both the erythroid and the myeloid lineages in vitro. The E26 genome contains two sets of cell-derived sequences, ets and myb. myb sequences are also present in avian myeloblastosis virus, which transforms myeloblasts exclusively. To determine whether the ets sequence is responsible for the erythroid specificity of E26, we analyzed the transforming activities of several viruses carrying mutations in the ets sequence constructed in vitro. The mutant viruses retained the ability to transform myeloid cells in vitro, indicating that the myb oncogene is sufficient for this viral function. However, the ets-deficient viruses did not cause an overt leukemia in chickens. The results indicate that the ets sequence is required for the induction of erythroblastosis by E26. of chicken cellular DNA which has been inserted into the pBR328 plasmid vector (18). The helper virus clone used in these studies was a A bacteriophage clone of myeloblastosisassociated virus type 1 (MAV-1) provided by M. Baluda (University of California, Los Angeles) (19). fs2 was constructed by filling in the unique BgJII restriction endonuclease site in pE26 by using the Klenow fragment of DNA polymerase and T4 DNA ligase. This created a ClaI restriction site in the fs2 clone. A4 and A18 were generated by partially digesting pE26 with endonuclease PstI, ligating the digested molecules, and selecting plasmids with the appropriate size deletion. Generation of infectious viruses. A 10-,ug sample of plasmid DNA was transfected onto 3 x 106 secondary chicken embryo fibroblasts on a 10-cm dish as a calcium phosphate precipitate (11) together with 1 ,ug of the K MAV-1 DNA. The day after transfection, the fibroblasts were transferred 1: 3 onto a new 10-cm dish and cocultivated for 24 h with chick bone marrow leukocytes which had been purified over Ficoll-Paque (Pharmacia). The nonadherent cells were then removed and grown in suspension in a myeloid cell growth medium containing 15% fetal calf serum, 5% heat-inactivated chicken serum, nonessential amino acids (GIBCO) with an additional 30 ,ug of L-asparagine per ml, 10-4 M a-thioglycerol, and 0.7% concanavalin A-stimulated chick spleen cell-conditioned medium (1) in Dulbecco modified Eagle medium. Within 7 to 10 days, a population of transformed cells predominated, and the tissue culture supernatants of these cells were used as virus stocks. Titers of viruses were determined by endpoint dilution to quantify the ability to form transformed colonies of bone marrow cells suspended in myeloid cell growth medium containing 1% methylcellulose. Analysis of viral gene products. Transformed myeloblasts (2 x 106) were labeled for 2 to 4 h with [35S]methionine (50 ,uCi/ml) in methionine-free Dulbecco modified Eagle medium supplemented with 0.7% spleen cell-conditioned medium (1) after a 45-min starvation in methionine-free Dulbecco modified Eagle medium. The cells were lysed in radioimmunoprecipitation assay buffer, the lysates were clarified, and the proteins were immunoprecipitated as described previously (22). The antiserum used for immunoprecipitation, directed against a myb-specific peptide, was obtained from M. Baluda

Avian retrovirus E26, originally identified as an erythroblastosis virus (12), induces the proliferation of both infected erythroid and myeloid blast cells (16, 20). The viral genome encodes a protein of Mr 135,000 (P135) which derives from several genetic elements: the retroviral gag gene (272 codons), the chicken c-myb gene (283 codons), and additional chicken sequences comprising what has been termed v-ets (491 codons) (13, 17, 18). The first 75 codons of the v-ets sequence are found in the chicken genome as exonlike sequences which map some 40 kilobases upstream of the main c-ets transcription unit (8, 10). The next 400 v-ets codons are derived from residues 28 to 428 of the chicken c-ets-1 gene, with three single-amino-acid changes (4, 6, 8, 10). The final 16 codons of v-ets are derived from DNA sequences with an unknown origin and replace the Cterminal 13 codons of the otherwise highly conserved c-ets sequence (4, 6, 8, 10). myb was initially identified as the oncogenic element of avian myeloblastosis virus (AMV), which transforms myeloid cells exclusively (20). In vitro assays of E26 virus on bone marrow and blastoderm cells indicate that E26 can transform erythroid cells and bipotent erythroid-myeloid precursor cells in addition to more mature cells of the myeloid lineage (16). Mutants of E26 that are temperature sensitive for myeloblast transformation, but not for the transformation of erythroid cells, have been isolated (2). These data suggest that the v-ets sequence might function primarily in the transformation of cells of the erythroid lineage. In addition to having a potential role in avian leukemia, sequences related to ets have been found translocated in several human nonlymphocytic leukemias (7, 21). To determine whether ets might have an oncogenic function, mutants of E26 virus deficient in v-ets were made and assayed for their transforming potential. MATERIALS AND METHODS Recombinant DNAs. The wild-type E26 clone used here, termed pE26, is a complete provirus flanked by short regions * Corresponding author. t Present address: Pharmacia Genetic Engineering, Inc., La Jolla,

CA 92037.

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ets IN AVIAN ERYTHROBLASTOSIS

n"E

Agag E26

ets

A env

TRANSFORMATION leukemia myeloblasts in vivo in vitro ++

;13-

399

+

P135

+ 4bp I

fs2

T +

-1266bp

A4

I

I

_I +

- 874bp

A18

i

FIG. 1. Structures of the wild-type E26 and mutant viral genomes and their gene products. The relative abilities of the viruses to transform no myeloid cells in vitro and cause erythroblastosis in vivo are indicated (+ +, high degree of transformation; +, transformation; and transformation). -,

(5) and precipitation was competed by the addition of 1 ,ug of the synthetic myb peptide 2 initially used as an immunogen. The immunoprecipitated proteins were analyzed by electrophoresis on 7.5% polyacrylamide-0.17% bisacrylamide gels containing 0.1% sodium dodecyl sulfate. The gels were impregnated with diphenyloxazole and exposed to presensitized Kodak XAR film at -70°C for 3 to 7 days. In vivo pathogenicity. The ability of viruses to induce erythroleukemia was tested by inoculating 1-day-old SPAFAS C/O chicks intramuscularly in the leg with 50 to 7,800 CFU of virus in 0.1 ml of medium. The induction of leukemia was monitored by noting the presence of large immature blast cells in blood smears. The erythroid nature of the blasts was tested by benzidine staining. Chicks were also inoculated intravenously in ovo at day 12, and cultures of bone marrow cells were initiated 20 days after hatching. RESULTS To initiate a mutational analysis of the E26 genome, it was necessary to identify a protocol for generating infectious virus from the pE26 DNA clone. Transfection of chicken embryo fibroblast cells with E26 and MAV-1 DNAs did not generate cultures that could stably produce a sufficient titer of E26 virus to transform bone marrow cells or infect chickens (data not shown). Since E26 does not transform chick fibroblasts, the lack of a selection for E26-infected cells presumably led to the overwhelming spread of the helper virus. Moreover, although transfection of pE26 DNA into rat fibroblast cell line 208F together with the selectable marker for neomycin resistance gave rise to cell clones that contained the intact E26 provirus, the P135 gene product was still not expressed (data not shown). These results suggest that E26 is not expressed efficiently in chicken or rat fibroblasts. Virus stocks with titers greater than 104 myeloblast CFU (MCFU) per ml generate fewer than 103 transformed cell clones in 106 bone marrow leukocytes (M. Nunn, personal observations), indicating that there is a limited population of

target cells for E26 in the marrow. Therefore, even if transfection of DNA directly into bone marrow cells were efficient, transfecting E26 DNA into these cells would not be expected to lead to efficient myeloblast transformation. To circumvent the problems of expression, transfection efficiency, and target specificity, the following protocol for the generation of infectious E26 virus was developed. Chicken embryo fibroblasts were transfected with pE26 and X MAV-1 DNAs and after 24 h were cocultivated with chick bone marrow cells to allow infection with virus being expressed transiently. After incubation for 24 h, the nonadherent bone marrow cell population was removed from the culture and grown in a rich medium supplemented with chick myelomonocytic growth factor (1). In this medium, cells which resembled E26- or AMV-transformed myeloblasts gradually predominated in the culture. Transformed myeloblasts are the only hematopoietic cells that grow extensively under these culture conditions (16, 20), and such cells did not arise from control cultures in which MAV-1 DNA alone was transfected. Infectious virus produced by the transformed myeloblasts was used for subsequent assays. Various insertion and deletion mutants of E26 in v-ets were constructed and are diagrammed in Fig. 1. fs2 carries a 4-base-pair insertion at the BglII restriction site in v-ets at sequence position 1063 (17). This mutation leads to early termination of the wild-type P135 protein at a nonsense codon 30 codons downstream of the BgJII site, which is 178 amino acids into the 491-amino-acid v-ets sequence. A4 carries a deletion of 1,266 base pairs from the PstI site at v-ets sequence position 663 to the PstI site at position 1929 (17). This deletion leaves sequences encoding 43 amino acids of the N-terminal portion of the v-ets region and links them, in the original reading frame, to the 26 codons encoding the C terminus of the v-ets sequence. A18 carries a deletion from the PstI site at v-ets sequence position 1929 to a PstI site in env sequences approximately 874 base pairs downstream. This mutation removes the coding region for the C-terminal 26 amino acids of v-ets and replaces them with the coding region for the last 125 amino acids of gp37env. An additional

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A.E26

CA4

B.fs2

FIG. 2. Myeloblasts transformed by wild-type E26 (A) and the ets-deficient fs2 (B) and A4 (C) mutant E26 viruses. Original magnification, x306.

mutant, Amyb, was generated by site-directed mutagenesis to eliminate the myb region of E26 from sequence positions -315 to 534 (18), fusing the gag region to the open reading frame of ets (confirmed by DNA sequencing). Upon transfection and cultivation of cells as described above, the fs2, A4, and A18 DNAs generated viruses that could transform myeloblastic cells. The Amyb plasmid did not give rise to transformed cells by these techniques. These results indicate that the ets sequence is not required for the transformation of myeloid cells by E26. Initially, the population of cells transformed by fs2 and A4 was somewhat less viable and morphologically more heterogeneous than cells transformed by wild-type E26 virus. The fs2- and A4-infected myeloblasts appeared somewhat crenulated with extending processes, and many more cells were flattened and adherent than with wild-type-infected cells (Fig. 2). To test the growth and morphology of the transformed cells in a different way, bone marrow cells were seeded into myeloid growth medium containing 1% methylcellulose immediately after infection with virus stocks or after cocultivation with transfected cells as described above (16, 20). Colonies of transformed cells could be seen after 7 days. The sizes and morphologies of the myeloblast colonies produced by infection with fs2, A4, A18, or wild-type E26 virus were indistinguishable (data not shown). No colonies of transformed myeloblasts were seen in uninfected or MAV-1-infected cultures. To determine whether myb-containing proteins of the expected sizes were present in the infected cells, proteins were immunoprecipitated from [35S]methionine-labeled myeloblasts by using an anti-myb peptide antiserum (5). P135 precipitated from wild-type E26/E26-associated virustransformed myeloblasts isolated from an infected chicken (Fig. 3, lane 1) comigrated with P135 precipitated from E26/ MAV-1-transformed cells generated as described above (lane 2; the 110,000-Mr protein is presumed to be a degradation product of P135, which is not precipitated with anti-gag

serum [M. Nunn, unpublished observations]). A series of proteins of Mr 102,000 to 105,000 were precipitated from fs2-infected cells (lane 3). A myb-containing protein was precipitated from A18-infected cells (lane 5), which is slightly larger than P135 from E26/MAV-1-transformed cells (lane 4). myb-specific proteins of Mr 77,000 to 79,000 were precipE26

wt I

+

fs2

E26 aM8 A4 4 + 5 + 6 +

2 + 3 +

N

P135-

-

P135- tof -

S

-

X

-fs2

9668-

as _-

a

68p

-_

4242-

FIG. 3. Electrophoretic analysis of the transforming proteins encoded by wild-type and mutant E26 viruses. [35S]methioninelabeled myeloblast lysates were immunoprecipitated with an antimyb peptide antiserum (1) either alone or with the addition of 1 ,ug of competing peptide (+ lanes), as described in Materials and Methods. The myeloblasts were transformed by a wild-type (wt) E26 isolate (lane 1), wild-type E26 virus derived from the pE26 clone (lanes 2 and 4), fs2 (lane 3), A18 (lane 5), or A4 (lane 6). The migrations of molecular weight markers in parallel lanes are indicated, with the marker sizes given in kilodaltons.

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TABLE 1. In vivo leukemogenesis of wild-type and mutant E26 viruses No. of chicks with

Latency to

Virus

erythroblastosisa (leukemic/total)

death (days)

E26/MAV-1 fs2/MAV-1 A4/MAV-1 A18/MAV-1

5/5 3/9 0/10

23-28 38, 40, and 54

0/7

a The number of the total animals injected which died with erythroblastosis. The remaining animals were maintained for 74 to 118 days without evidence of illness (except as noted in the text).

itated from A4-infected cells (lane 6). The myb-related products of fs2, A4, and A18 were of the approximate Mrs expected for the predicted proteins of 763, 624, and 1,145 amino acids, respectively, given an apparent Mr of 135,000 for the 1,046-amino-acid product of wild-type E26. In each case, the immunoprecipitation of the myb-related protein was blocked by the addition of excess myb peptide to the reaction (Fig. 3, + lanes). The approximately 54,000-Mr species blocked by the addition of peptide in all samples is not myb related (5). The detection of multiple myb-related proteins in the mutant-infected cells suggests that the proteins are modified subsequent to their translation. P135 is also modified, although the various modified forms are not always resolved by electrophoretic analysis (3). Under the labeling conditions used here, the mutant proteins appear to accumulate to similar levels as wild-type P135, suggesting a similar synthesis rate and stability. Since ets sequences are not essential for transformation of myeloid cells, the ability of these viruses to transform erythroid cells was tested. Unfortunately, in vitro assays for E26-transfortned erythroblasts have not been routinely successful, requiring the use of an in vivo assay for erythroblastosis. The SPAFAS chicks used here are susceptible to E26-induced erythroblastosis but are resistant to AMVinduced myeloblastosis (M. Nunn, unpublished results). Also, AMV stocks with a MAV-1 pseudotype, the helper virus used in these experiments, are known to be less acutely oncogenic than other pseudotypes (14, 15). Thus, the combined use of SPAFAS animals and the MAV-1 helper virus provides a clear distinction between the abilities to transform erythroid and myeloid cells. One-day-old chicks were injected with at least 50 MCFU of wild-type or mutant virus. Wild-type virus (as little as 60 MCFU) induced an erythroblastic leukemia and killed the animals within 28 days (Table 1). The disease was characterized by anemia, hepatosplenomegaly, and the presence of immature blast cells in the blood. The blasts were of the erythroid lineage, as determined by a weak benzidine-positive staining. Unlike AMVor E26-transformed myeloblasts, these erythroid cells were unable to grow in vitro in myeloid growth medium. In contrast to animals infected with wild-type E26 virus, none of the animals infected with A4 or A18 became leukemic, with viral inocula as high as 4,450 MCFU. fs2 caused -an erythroblastic leukemia in three of nine animals with a longer latency (38, 40, and 54 days) than that caused by wild-type E26. However, when leukemic blast cells were isolated from two of these leukemic fs2-infected animals, they were found to synthesize a wild-type P135 and virus which caused an erythroleukemia of wild-type latency in vivo (data not shown). This indicates either that a reversion of the frameshift mutation had occurred in these animals or that a low

401

level of wild-type virus had contaminated the initial fs2 inoculum. We conclude that the fs2 mutant itself is also not erythroleukemogenic. A total of 2 of the 10 A4-infected birds and 2 of the 9 fs2-infected animals died of a severe anemia at 27 to 44 days postinoculation. This may have been due to MAV-1 helper virus infection (23). Chicks inoculated intravenously in ovo at day 12 with 100 MCFU of the A4 mutant virus also did not develop erythroblastosis. The buffy coat cells of two of these animals 20 days after hatching were cultured in myeloid growth medium, and within 7 to 12 days rapidly proliferating myeloblasts appeared in the cultures, indicating that the mutant virus retains the ability to transform myeloid cells in vivo as well as in vitro.

DISCUSSION These results show that the ets region is required for E26 to transform erythroblasts. Although competent for transformation of myeloid cells, none of the viruses with mutations in v-ets could cause erythroblastosis. The inactivation of ets function by the A18 mutation is particularly interesting, because this mutation removes only the C-terminal 26 amino acids of the ets sequence. The viral ets sequence diverges from both the normal chicken c-ets gene and the human c-ets-2 gene 16 amino acids from the v-ets C terminus (4, 6, 8, 10, 24), which suggests that structural alterations in this region of the c-ets sequence played a role in the generation of the leukemogenic activity of the viral oncogene. Our data show that the placement of foreign sequences in this Cterminal region of the v-ets sequence destroys its leukemogenic function. The ets-deficient viruses retained the ability to transform myeloid cells in vitro, indicating that the myb gene of E26 can function independently of the ets gene, in a fashion analogous to that of the myb gene of AMV. Although these viruses did not cause a myeloid leukemia in this study, there are infected myeloid cells in some animals that can proliferate under the in vitro tissue culture conditions used here. The use of appropriate helper viruses and hosts might lead to generation of an AMV-type hyperplasia of myeloid cells in vivo. Although it is shown here that v-ets is required for the induction of erythroblastosis by E26, it remains to be determined whether the v-ets sequences are sufficient to transform erythroid cells in the absence of the myb oncogene. Experiments with the Amyb mutant, which lacks all of the E26 myb sequence but retains v-ets, indicate that v-ets alone cannot transform myeloid cells, but we have not yet been able to test whether the Amyb mutant induces erythroblastOsIs.

Cellular ets sequences have been shown to be translocated in several human nonlymphocytic leukemias (7, 21). The rearrangement of ets sequences may have been the primary event in the generation of these diseases; however, the simplest interpretation of the data presented here is that the v-ets sequence is modulating myb function as an enhancer of leukemogenicity in E26, rather than acting as a dominant oncogene. In this regard, E26 would be similar to other avian retroviruses which carry multiple cell-derived sequences. In the ES4 strain of avian erythroblastosis virus, the erbA sequence has been shown to be dispensable, with the erbB oncogene providing the primary transforming function (9), and in MH2 virus the mil sequence is not required for the transformation of cells in vitro, with the myc oncogene providing the dominant role (25). In each case, a replication-

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competent retrovirus appears to have captured a multistage leukemogenic event in process and incorporated the essential genetic elements into an acutely oncogenic virus. ACKNOWLEDGMENTS We thank T. Graf and co-workers for performing the inoculation and evaluation of chicken embryos described above, and W. J. Boyle, J. Lipsick, and M. Baluda for providing the MAV-1 DNA clone and the myb antiserum used here. We also thank P. Duesberg for providing support for the initial stages of this work and thank him and L. Tack, J. Lipsick, K. Radke, C. Moscovici, and T. Graf for advice and discussions. This research was supported by Public Health Service grants CA17096 (T.H.), CA28458 (T.H.), CA39780 (T.H.), and CA11426 (P. Duesberg, University of California, Berkeley) from the National Cancer Institute, and by grant 1547 (P.D.) from The Council for Tobacco Research-USA, Inc. M.N. was a Leukemia Society of America Special Fellow.

11.

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