The Molecular Biology of the Myeloproliferative Leukemia Virus
F. Wendling 1, M. Souyri 2, I. Vigon 2, S. Gisselbrecht 2, and P. Tambourin 3    Hämatol. Bluttransf. Vol 35

1 Unite de Recherche en Genetique Moleculaire et en Hematologie, TNSERM U.91 CN RS U A 607, Hopital Henri Mondor , 94010 Creteil, France.
2 INSERM U 152, lnstitut Cochin de Genetique Moleculaire (ICGM), Hopital Cochin, 27 Rue du Faubourg Saint Jacques, 75014 Paris, France.
3 Institut Curie, 27 Rue d'Ulm, 75005 Paris, France.


Most acutely transforming leukemogenic or sarcomagenic retroviruses have transduced in their genomes altered cellular genes named oncogenes [1]. These viruses are usually defective in replication as a result of the deletion of structural genes, and they require a helper virus for propagation. Isolation of new acutely transforming retroviruses, comprehensive studies of their physiopathological processes, and molecular analysis of their genomes remain, therefore, powerful means for the identification of key genes involved in the regulation of cell growth, differentiation, or development. The purpose of this paper is to summarize the data that we have accumulated over the past few years on a recently isolated acute leukemogenic murine retrovirus named myeloproliferative leukemia virus (MPL V).

Isolation of the MPLV

We isolated the MPL V in 1985 at the Curie Institute (Orsay, France) during a research program designed to evaluate the in vivo transforming properties of different Friend helper viruses (F MuL V). While several clonal F-MuL V isolates have the capacity to induce a rapid erythroblastosis in newborninoculated NIH Swiss or BALB/c mice [2], DBA/2 mice were found to be resistant to this early erythroleukemia [3]. Nevertheless, they developed various types of hematopoietic malignancies after a latent interval of712 months [4-6]. In general, these leukemias ( either myelogenous, lymphoid, or erythroid) were associated with a more or less severe anemia. Out of 238 DBA/2 mice inoculated at birth with F -MuL V clone 57 [7], one mouse developed, after 7 months of infection, an hepatosplenomegaly unusually accompanied with a polycythemia. Cellfree extract prepared from the original leukemic spleen or supernatant medium from an in vitro permanent cell line derived from the leukemic spleen cells caused an explosive leukemia upon inoculation into adult mice of most strains, including C 57 Bl strains. The disease was characterized by hepa tosplenomegaly, polycythemia, pronounced myelemia but no thymus or lymph node involvement, and death within 1- 3 months. Spleen and liver were extensively infiltrated with maturing precursor cells belonging to the granulocytic, erythroblastic and megakaryocytic lineages. Typically, the blood of severely diseased animals was also massively invaded by morphologically normal polymorphonuclears, erythroblasts, and platelets. Several hematopoietic lineages were obviously involved in this disease, hence our name for the virus isolate, "myeloproliferative leukemia virus" [8].

Genetic Analysis of MPL V Isolate

Virologic studies of MPL V by Penciolelli et al. [9] demonstrated that this highly leukemogenic virus isolate contained two dissociable retroviral genomes: one was the parental replication-competent FMuL V 57, and the second was a new replication-defective component now designated as MPL V. A comparison of viral RN A species expressed in F -MuLV alone or F-MuLV + MPLV-producing cells by Northern blot analysis showed that MPLV was 0.8 kb shorter than F-MuLV and that a deletion had probably occurred in the MPL V env gene. This was further confirmed by the establishment of the MPL V restriction endonuclease map which was compared with that of F-MuLV [10]. From their data, these investigators concluded that the MPLVdefective genome

(a) was derived from F-MuLV,
(b) had conserved the F -M uL V gag and pol regions, and
(c) was deleted and rearranged in its env region [9].

Although MPLV does not transform fibroblasts in culture, its isolation free of replicating F-MuLV in nonproducer cells was feasible since the MPL V titer in the original isolate was approximately equivalent to that of F-MuLV. By the technique of limiting dilution and singlecell cloning, nonproducer cells containing MPL V were derived from Mus dunni fibroblasts [9]. Supernatant medium from these nonproducer cells did not cause any disease in inoculated mice demonstrating the defectiveness of MPL V. However , when superinfected with a variety of replicating helper viruses, supernatants reproduced the same acute myeloproliferative syndrome as caused by the original isolate. These experiments provided circumstantial evidence that the helper-dependent MPL V genome contained the genetic information necessary for the observed pathological processes.

Genomic Composition of MPLV

In an attempt to define the origin and nature of the genetic sequences contained in the MPL V -rearranged env region, Souyri et al. [11] derived cDNA probes which were nonhomologous to sequences contained in F-MuLV. Two probes were found to be MPL V specific, in that they hybridized to RNA of MPLV -containing nonproducer cells but did not hybridize to RNA ofecotropic MuLVs norto RNA of amphotropic or xenotropic murine viruses. This indicated that, in contrast to Friend spleen focus-forming viruses (SFFV), MPL V did not result from a recombination between F-MuLV and a portion of the env gene of murine xenotropic virus [ 12, 13] . A full-length biologically active MPL V provirus was molecularly cloned from a genomic library of a nonproducer Mus dunni clone [11 ]. Sequence analysis revealed that the MPL V env gene contains a large open reading frame which could code for a polypeptide of 284 amino acids. This protein would contain 64 amino acids derived from the amino terminus of the F-MuL V gp70, including the signal peptide, 36 amino acids from a central region of the F-MuLV env gene, and 184 amino acids that are specific to MPL V (Fig. 1). A hydrophobicity plot of the amino acids sequence revealed that, in addition to the 34 hydrophobic amino acids of the gp 70 signal peptide, the MPL V -specific domain contained a stretch of 22 uncharged amino acids. Thus, the putative MPL V env product presents the features of a transmembrane protein comprising an extracellular domain of 143 amino acids, a single transmembrane domain of 22 amino acids, and a cytoplasmic domain of 119 amino acids without consensus sequence for kinase activity [14]. Computer analysis of the deduced amino acid sequence revealed that the MPL V -specific sequence did not correspond to any known genes.

Fig. I. Schematic representation of the putative MPLV env product. The ENV-mpl fusion protein consists of three fragments: part 1 derives from the NH2-terminal region of the F-MuLV envelope gene (gp70) and contains a signal peptide; part 2 derives from a central part of the F- MuLV gp 70; part 3 corresponds to a transduced cellular sequence, most probably truncated at its N-terminus. The product encoded by this rearranged gene has the features of a single mernbrane-spanning domain (TM). The extra cellular domain of v-mpl possesses the amino acid sequence WSAWS highly conserved in the hematopoietin receptor superfamily, while the cytoplasmic domain does not contain consensus sequence for know catalytic activity

MPLV Has Transduced a Novel Oncogene

Since nonviral sequences found in the genome of acutely transforming retroviruses derive from cellular genes and are conserved phylogenetically, we looked for the presence of MPLV -specific sequences in genomic DNA from different mammals. Under stringent hybridization conditions, discrete bands were revealed in DNAs from mouse, rat, mink, dog, cow, and human. In addition, MPLV specific probes recognized a 3.0-kb mRNA in spleen and bone marrow from adult mice and in fetal liver cells, but not in nonhematopoietic tissues [11 ]. Thus, taking into consideration the biological properties of MPL V, the cellular origin of the sequence contained in its env gene, the conservation in the genome of mammals and the expression in normal hematopoietic tissues, we concluded that MPL V had transduced a novel oncogene which was designated as v-mpl. By in situ hybridization and genetic analysis studies, chromosomal localization of the c-mpl proto-oncogene was assigned to mouse chromosome 4 (Vigon et al., unpublished data) and to human chromosome 1 p34 [15].

Leukemogenic Properties of MPLV

In vivo studies by Wendling and coworkers have indicated that MPL V induced a rapid suppression of growth factor requirements for in vitro colony formation of a large spectrum of committed as well as multipotential progenitor cells [16, 17]. The primary manifestation of viral infection was a switch to erythropoietin (EPO) independence of the colony forming unit-erythroid (CFU-E) population which was complete in the spleen after 6 days of infection. A possible stimulating effect of EPO present or secreted in the culture medium was ruled out by the addition of neutralizing antiEPO antibodies to the culture system. The effects of MPL V infection on the early and primitive erythroid progenitor cells (BFU-E) was assessed in methylcellulose serum-free cultures. It was found that well hemoglobinized pure and mixed erythroid colonies developed without the addition of interleukin-3 or EPO. Moreover, while a majority of colonies contained erythroblasts mixed with megakaryocytes, about 12% revealed three or more lineages of differentiation [16]. Further in vivo studies have documented that MPL V infection also induced the spontaneous colony formation of myeloid progenitors, i.e., granulocyte macrophage colony-forming cell (GMCFC) granulocyte (G)-CFC, mega karyocyte (Meg)-CFC, and mixed CFC, probably as a result of direct infection of these progenitors and not as a consequence of a paracrine secretion of soluble colony stimulating factors by the accessory cells [17]. These observations supported the conclusion that MPL V acts on various progenitors, inducing their proliferation and terminal differentiation independently of signals normally provided by colony stimulating factors, interleukins, EPO, or any conditioned medium. However, formal proof that MPL V can transform hematopoietic target cells in the absence of coinfection with a replicating MuL V was not provided by these experiments. We addressed this question by producing helper-free MPL V stocks using the packaging psi-CRE cell line that produces a high titer of infectious, nonreplicating particles but does not yield helper virus [18]. When adult ICFW mice were intravenously given helper-free preparations of MPL V, more than 90% of the mice were healthy 2 months after inoculation. Nevertheless, we observed that MPL V induced a mild but transient spleen enlargement with the appearance of colonies well visible on the spleen surface on days 5, 10, and 15 after inoculation. Histologically, colonies were composed of erythroblasts, or erythroblasts, granulocytes, and megakaryocytes clustered together in the splenic red pulp. On day 25 and thereafter, these colonies disappeared, leaving spleens with a normal aspect. In contrast, when helperfree preparations of MPL V were injected into mice pretreated with the aplastic drug 5- fluorouracil ( 5- FU, 150 mg/kg body weigth, 4 days before virus inoculation), all animals developed atypical MPL V syndrome and died from overt leukemia within 2 months (Wendling et al., unpublished data). Together these data indicate that

(a) the MPL V component is primarily responsible for the myeloproliferative effects of the viral complex,
(b) expression of MPL V in erythroid and myeloid progenitors abolishes their growth factor requirement for in vitro colony formation, and
(c) induction of leukemia occurs in 5FU -pretreated mice, suggesting that stable infection of cycling primitive progenitors is critical for leukemia development.

In Vitro Transformation Properties of MPLV

An area of current research in our laboratory is related to the ability of a helperfree preparation of MPL V to transform hematopoietic cells in vitro. A 2-h incubation of bone marrow cells enriched in highly dividing primitive progenitors by treatment of mice with 5- FU was sufficient to induce autonomous colony formation of about 30% of the colonyforming cells present in the preparation. Cytologically, half of these spontaneous colonies were composed of either granulocytes, megakaryocytes, or erythrocytes, while the remainders were mixed colonies of which about 20% contained three or more lineages of differentiation. Upon replating, the multilineage colonies produced secondary and tertiary mixed colonies, suggesting self-renewal [11]. The question of whether or not transformation of hematopoietic progenitors would lead to the generation of immortalized cell lines was then investigated. When marrow cells were cultured in liquid medium, it was observed that rapidly dividing nonadherent cell populations were produced in MPL V -infected cultures. After 10 to 12 days, these nonadherent populations could be transferred into fresh flasks devoid of stromal feeder layers. Cells continued to proliferate and generated permanent suspension cultures containing polymorphonuclears, megakaryocytes and erythroblasts. Upon continuous passages, the majority of the cell lines evolved towards a more restricted phenotype which remained stable over several months. Diverse immortalized megakaryocytic, myelomonocytic, erythroblastic, or mastocytic cell lines retaining the ability to differentiate could easily be obtained. Since these permanent cell lines evolved from a multipotential to a more restricted phenotype, we investigated whether they were polyclonal or monoclonal by studying proviral-cell DNAjunctions. Cultures were polyclonal 5 days after initiation. However, after 3 weeks and at a time where all cultures displayed a multipotential phenotype, one or a few major proliferating clones were detected in each cell line. Interestingly, the same clones were still found after 3 months of continuous passages when the cell lines appeared to be restricted in their differentiation potential [11]. Thus, it seems likely that MPLV induces the clonal outgrowth of a single or few transformed, probably multipotentiaI, stem cells (clonal selection), the full differentiation capabilities of which being lost along with continuous culturing (clonal evolution). The obtaining of immortalized in vitro cell lines raised the question of whether cells were tumorigenic. To approach this problem, 2 x 106 cells were subcutaneously grafted into either syngeneic or nude mice. Upon repeated assays, none of the cell lines developed tumor nodules at the site of inoculation when cells from cultures less than 4 months old were grafted. After prolonged passages (more than 7 months), 60% of the cell lines produced hematopoietic subcutaneous tumoral nodules, suggesting that additional genetic events must have occurred to reach a full malignant state.

Summary and Current Knowledge

The myeloproliferative leukemia virus isolate consists of two distinct viral components: a replicating F-MuLV and a helper-dependent MPL V. MPL V accounts for the rapid in vivo and in vitro transformation of a broad spectrum of multipotential, myeloid, and erythroid progenitors which acquire growth factor independent proliferation and differentiation. By sequence analysis of a biologically active clone, MPL V has been shown to be an env recombinant virus containing sequences derived from the F-MuL V env gene and additional nonviral cellular sequences. These nonviral sequences are conserved in various mammals and are expressed in hemopoietic tissues from normal mice. MPLV was thus generated by transduction of an oncogene (v-mpl) in the envelope region of an F -MuLV genome. v-mpl does not correspond to any known gene, but the putative MPL V env fusion product has the features of a transmembrane protein with the Nterminal signal sequence of the F-MuLV gp70 directing the polypeptide across the membrane and a single transmembrane domain. Interestingly, the extracellular domain of v-mpl possesses, 13 amino acids upstream to the membranespanning domain, the amino acid sequence WSXWS, highly conserved in all cy to kine receptors that make up the hematopoietin receptor superfamily [19]. In addition, a significant number of conserved amino acids were found when the extracellular domain ofv-mplwas aligned with that of the IL-2ß, IL-3, IL-4, IL-6, IL- 7, GM-CSF, G-CSF, and EPO receptors [11]. Since the N-terminal part of the fusion protein consists of F -M uL V derived sequences, it is not yet known whether the c-mpl proto-oncogene product would contain the highly conserved cysteine residues characteristically found in the ligand-binding domain of each of these receptors [ 19] .Nevertheless, with regard to the general features of v-mpr, it is tempting to speculate that MPL V has transduced a truncated form of a putative cytokine receptor. Cloning of the protooncogene cDNA is currently underway in our laboratory to allow further comparison. A major focus of future research will be to understand the mechanism by which this viral oncogene can short-circuit the growth-regulatory signals delivered by the binding of various hematopoietic growth factors to their specific receptors. This requires further studies on the mechanism of signal transduction by MPL V and by other receptors of the same family.


We thank Martine Charon, Laurence Cocault and Paule Varlet who provided excellent technical assistance. This work was supported by grants from the Institut National de la Recherche Medicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), the Association pour la Recherche contre la Cancer (ARC), the Fondation pour la Recherche Medicale, and the Ministere de la Recherche et de la Technologie.


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