Acute leukemia viruses E26 and avian myeloblastosis

Replication-defective acute leukemia viruses E26 and myeloblastosis virus (AMV) cause distinct leukemias although they belong to the same subgroup of oncogenic avian tumor viruses based on shared transformation-specific (onc) RNA sequences. E26 causes predominantly erythroblastosis in chicken and in quail, whereas AMV induces a myeloid leukemia. However, upon cultivation in vitro for >1 month, a majority of surviving hemopoietic cells of E26-infected animals bear myeloid markers similar to those of AMV-transformed cells. We have analyzed the genetic structure and gene products ofE26 virus for a comparison with those of AMV. An E26/helper virus complex was found to contain two RNA species: a 5.7-kilobase (kb) RNA that hybridizes with cloned AMV-specific proviral DNA and hence is probably the E26 genome; and an 8.5-kb RNA that is unrelated to AMV and represents helper virus RNA. Thus, E26 RNA is smaller than 7.5kb AMV RNA. Hybridization of size-selected poly(A)-terminating E26 RNA fragments with AMV-specific DNA indicated that the shared specific sequences are located in the 5' half of the E26 genome as opposed to a 3' location in AMV RNA. In nonproducer cells transformed in vitro by E26, a gag-related nonstructural 135,000-dalton protein (p135) was found. No gag (Pr76) or gag-pol (Prl80) precursors of essential virion proteins, which are present in AMV nonproducer cells, were observed. pl35 was also found in cultured E26 virus producing cells of several leukemic chickens, and its intracellular concentration relative to that of the essential virion proteins encoded by the helper virus correlates with the ratio of E26 to helper RNA in virions released by these cells. p135 is phosphorylated but not glycosylated; antigenically it is not related to the pol or env gene products. It appears to be coded for by a partial gag gene and by E26-specific RNA sequences, presumably including those shared with AMV. Hence, AMV and E26 appear to use different strategies for the expression of related onc sequences: AMV is thought to encode a transforming protein via a subgenomic mRNA, whereas E26 codes for a gag-related polyprotein via genomic RNA. It is speculated that differences in the oncogenic properties of E26 and AMV are due to differences in their genetic structures and gene products. E26, a replication-defective avian acute leukemia virus isolated in Bulgaria in 1962 from a fieldcase of erythroblastosis in a chicken, was initially classified as an erythroblastosis (E) virus (1). Subsequently, on the basis of in vitro transformation assays and in vivo studies it was proposed to reclassify E26 as a myeloblastosis virus (2-4). However, recent analyses of differentiation parameters of leukemic cells from chicken and quail clearly indicated that, in vivo, the primary hematopoietic target cell for E26 belongs to the erythroid lineage but also includes some myeloid cells (5, 6). This directly confirmed earlier reports that E26 induces the proliferation of predominantly erythroid cells in various avian species including chicken, turkey, guinea fowl, and Japanese quail (1, 7, 8). It has also recently been reported that E26 can transform cultured quail fibroblasts (4), but this has not been confirmed in other laboratories (unpublished data). Acutely transforming retroviruses of the avian leukosis/sarcoma group can be classified into seven subgroups on the basis of helper virus-unrelated, transformation-specific sequences present in the genomic viral RNA (9-11). Four subgroups of avian sarcoma viruses and three subgroups of acute leukemia viruses have been distinguished to date. On this basis, E26 and avian myeloblastosis virus (AMV) were classified as members of the same subgroup of acute leukemia viruses (9, 12-14). Different members of a given oncogenic subgroup regularly have similar genetic structures and induce similar forms ofneoplasia in the animal (9-11). However, the oncogenic spectra of E26 and AMV are distinct: cloned stocks of AMV exclusively induce myeloblastic leukemia, whereas E26 causes erythroid or mixed erythroid and myeloid leukemia (6,15). Both viruses have in common that they do not appear to transform fibroblasts. Additional oncogenic properties reported for the original isolate of AMV may reflect activities of the associated helper viruses (16). In the present communication we demonstrate that, despite the homology between their specific RNA sequences, the genetic structure and gene products of E26 RNA differ significantly from the structure and protein products of AMV RNA reported recently (12, 17). These differences are consistent with the distinct oncogenic properties of these two viruses. MATERIALS AND METHODS Cells and Viruses. E26 [E26-associated virus (E26AV)] was originally obtained from A. Therwath and K. Scherrer (Paris). Erythroblastosis was induced by intravenous injection of 0.1 ml of undiluted E26 virus stock (i.e., 102 focus-forming units, determined in a yolk sac culture assay) into 1-day-old SPAFAS (Storrs, CT) chickens. Blood smears were prepared twice weekly, and leukemia was usually apparent after 1-2 weeks. Blood was obtained by heart puncture, heparinized, and centrifuged for separation of blood cells. Cells from the buffy coat were cultured in vitro as described (12). In vitro transformation Abbreviations: NaCl/Cit, standard saline citrate (0.15 M NaCl/0.015 M sodium citrate, pH 7); kb, kilobase(s); RPV, ring-necked pheasant virus. t Present address: Max-Planck-Institut fur Molekulare Genetik, Ihnestrasse 63-73, D-1000 Berlin 33, Federal Republic of Germany. 3677 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 79 (1982) of chicken bone marrow cells by infection with E26 at low multiplicity generated virus-producing and some nonproducing cell colonies in agar or methocel suspension culture (18). Viruses used as sources for marker RNAs were: avian myelocytomatosis virus MC29'(MCAV)-I (19), MC29 deletion mutant viruses MC29-1OC and MC29-10H as pseudotypes with ring-necked pheasant virus (RPV) (20), and MAV-2 (12). Analysis of Viral'RNA. Radiolabeling, purification, and polyacrylamide gel electrophoresis of viral RNA were done essentially as described (12, 19-21). Hybridization ofviral RNA, fractionated by preparative gel electrophoresis (21) or gradient sedimentation (10, 22), with AMV-specific proviral DNA (23) was carried out as follows. RNA samples were suspended in 25 ,1. of H20 and dotted onto 20x NaCl/Cit-treated (1x NaCl/ Cit is 0.15 M NaCl/0.015 M Na citrate, pH 7) nitrocellulose sheets (Schleicher & Schuell), baked, and washed as described (24). Prehybridization (20 hr) and hybridization (20 hr) offilters was carried out at 680C in 20 ml of 6X NaCl/Cit containing 0.1% NaDodSO4, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, and 0.2% bovine serum albumin. A pBR322 plasmid containing the 1-kilobase (kb) Hae II-Xba fragment of proviral AMV DNA, which represents most of the helper virus-unrelated transformation-specific sequence ofAMV (23), was used here as a probe ofshared AMVand E26-specific RNA sequences (12). Plasmid DNA was labeled by nick-translation (25) to a specific activity of 6 x 107 cpm/Ig ofDNA by using [a-32P]dCTP (Amersham) with a specific activity of 410 Ci/mmol (1 Ci = 3.7 X 1010 becquerels). The probe was added to the hybridization mixture after denaturation at 95°C for 5 min. After hybridization, filters were washed four times with 2X NaCl/Cit containing 0.1% NaDodSO4 at 20°C for 5 min and then two times with 0.1 X NaCl/Cit containing 0.1% NaDodSO4 at 68°C for 30 min. Dots were visualized by autoradiography and quantitated by scintillation counting. Analysis of Virus-Specific Protein Synthesis. Labeling of cells with [3S]methionine, H332P04, or a mixture of [3H]glucosamine, [3H]mannose, and [3H]fucose, preparation of cell lysates, immunoprecipitation, antisera, and NaDodS04polyacrylamide gel electrophoresis have been described (10, 12, 21, 26, 27), with the addition here of 2% dialyzed calf serum to the A labeling medium intended to limit adherence of virus to infected cells.