Fli-1 overexpression in erythroleukemic cells promotes erythroid de-differentiation while Spi-1/PU.1 exerts the opposite effect.

The ETS transcription factors play a critical role during hematopoiesis. In F-MuLV-induced erythroleukemia, Fli­1 insertional activation producing high expression of this transcription factor required to promote proliferation. How deregulated Fli­1 expression alters the balance between erythroid differentiation and proliferation is unknown. To address this issue, we exogenously overexpressed Fli­1 in an erythroleukemic cell harboring activation of spi­1/PU.1, another ETS gene involved in erythroleukemogenesis. While the proliferation in culture remains unaffected, Fli­1 overexpression imparts morphological and immunohistochemical characteristics of immature erythroid progenitors. Fli­1 overexpression in erythroleukemic cells increased the numbers of erythroid colonies on methylcellulose and reduced tumorigenicity as evidenced by increase latency of erythroleukemogenesis in mice inoculated with these cells. Although all transplanted mice developed enlargement of the spleen and liver due to leukemic infiltration, Fli­1 overexpression altered the hematopoietic phenotype, significantly increasing the expression of regulatory hematopoietic genes cKIT, SCA-1, CD41 and CD71. In contrast, expression of Spi­1/PU.1 in a Fli­1 producing erythroleukemia cell line in which fli­1 is activated, resulted in increased proliferation through activation of growth promoting proteins MAPK, AKT, cMYC and JAK2. Importantly, these progenitors express high levels of markers such as CD71 and TER119 associated with more mature erythroid cells. Thus, Fli­1 overexpression induces a de-differentiation program by reverting CFU-E to BFU-E erythroid progenitor activity, while Spi­1/PU.1 promoting maturation from BFU-E to CFU-E.

Reduced Competitive Repopulation Capacity of Multipotential Hematopoietic Stem Cells in the Bone Marrow of Friend Virus-infected Fv2-resistant Mice.

BACKGROUND/AIM: The polycythemia form of Friend leukemia virus (FVP) causes splenomegaly and lethal erythroleukemia in Fv-2s-susceptible mouse strains. We sought to determine whether the hematopoietic stem cell (HSC) pool was expanded in Fv-2r-resistant mice. MATERIALS AND METHODS: The 120-day bone marrow transplantation competitive repopulation assay was used to determine whether FVP-infected Fv-2r C57BL/6 mice demonstrated expansion of the HSC pool compared to the pool of committed hematopoietic progenitor cells in the same marrow assayed in vitro. RESULTS: There was a significant expansion of committed hematopoietic progenitors observed in virus-infected Fv-2s FVB mice, but not Fv-2r C57BL/6 mice. Furthermore, Fv-2r mice showed no detectable expansion of either committed hematopoietic progenitor cells or the multipotential stem cell pool by competitive repopulation assay. CONCLUSION: Friend virus disease in Fv-2s mice is associated with expansion of committed hematopoietic progenitors. Fv-2r mice show no expansion of either committed progenitor or pluripotential stem cell numbers.

Differential requirements of cellular and humoral immune responses for Fv2-associated resistance to erythroleukemia and for regulation of retrovirus-induced myeloid leukemia development.

To assess the possible contribution of host immune responses to the exertion of Fv2-associated resistance to Friend virus (FV)-induced disease development, we inoculated C57BL/6 (B6) mice that lacked various subsets of lymphocytes with FV containing no lactate dehydrogenase-elevating virus. Fv2(r) B6 mice lacking CD4(+) T cells developed early polycythemia and fatal erythroleukemia, while B6 mice lacking CD8(+) T cells remained resistant. Erythroid progenitor cells infected with spleen focus-forming virus (SFFV) were eliminated, and no polycythemia was observed in B cell-deficient B6 mice, but they later developed myeloid leukemia associated with oligoclonal integration of ecotropic Friend murine leukemia virus. Additional depletion of natural killer and/or CD8(+) T cells from B cell-deficient B6 mice resulted in the expansion of SFFV proviruses and the development of polycythemia, indicating that SFFV-infected erythroid cells are not only restricted in their growth but are actively eliminated in Fv2(r) mice through cellular immune responses.

Novel cytotoxic exhibition mode of antimicrobial peptide anoplin in MEL cells, the cell line of murine Friend leukemia virus-induced leukemic cells.

Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α-helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus-induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose-dependent and time-dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G0/G1 phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent.

Avian leukosis virus subgroup J associated with the outbreak of erythroblastosis in chickens in China.

BACKGROUND: Emaciation, depression and lethargy were observed in two flocks of Chinese local breed and one flock of commercial layer chicken infected naturally from 2010 to 2011. The aims of this study were to diagnose. METHODS AND RESULTS: Gross observation showed that severe enlargement of liver, spleen and kidney, and hemorrhage of thymus, muscle and glandular stomach in all submitted birds. The liver and lung of one flock had diffuse, multifocal white raised foci on the surface as well as on the cut-surface. Numerous erythrocytoblasts with bigger volume, basophilic cytoplasm and round nucleus were observed in blood and bone marrow smears. The same erythrocytoblasts were also found crowded in blood vessels and mesenchym of tissues by histological examination, and some had mitotic figures. PCR results showed that three flocks were positive for ALV-J with specific fragment of 924 bp, negative for AEV, ALV-A, ALV-B, Marek's disease virus (MDV) and Reticuloendotheliosis virus (REV). The results of immunohistochemistry showed that cytoplasm of histiocytes and erythrocytoblasts in lung and spleen sections was positive for ALV-J antigen. CONCLUSION: These data demonstrated that erythroblastosis was all induced by ALV-J in the three different flocks. This is the first document report of erythroblastosis induced by ALV-J in China flocks.

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.

Self-renewal of leukemia stem cells in Friend virus-induced erythroleukemia requires proviral insertional activation of Spi1 and hedgehog signaling but not mutation of p53.

Friend virus induces erythroleukemia through a characteristic two-stage progression. The prevailing model proposes that during the initial, polyclonal stage of disease most of the infected cells terminally differentiate, resulting in acute erythrocytosis. In the late stage of disease, a clonal leukemia develops through the acquisition of new mutations--proviral insertional activation of Spi1/Pu.1 and mutation of p53. Previous work from our laboratory demonstrated that Friend virus activates the bone morphogenic protein 4 (BMP4)-dependent stress erythropoiesis pathway, which leads to the rapid expansion of stress erythroid progenitors, which are the targets for Friend virus in the spleen. We recently showed that stress erythroid progenitors have intrinsic self-renewal ability and therefore could function as leukemia stem cells (LSCs) when infected with Friend virus. Here, we show that the two stages of Friend virus-induced disease are caused by infection of distinct stress progenitor populations in the spleen. The development of leukemia relies on the ability of the virus to hijack the intrinsic self-renewal capability of stress erythroid progenitors leading to the generation of LSCs. Two signals are required for the self-renewal of Friend virus LSCs proviral insertional activation of Spi1/Pu.1 and Hedgehog-dependent signaling. Surprisingly, mutation of p53 is not observed in LSCs. These data establish a new model for Friend virus-induced erythroleukemia and demonstrate the utility of Friend virus as a model system to study LSC self-renewal.

The inositol phosphatase SHIP-1 is negatively regulated by Fli-1 and its loss accelerates leukemogenesis.

The activation of Fli-1, an Ets transcription factor, is the critical genetic event in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. Fli-1 overexpression leads to erythropoietin-dependent erythroblast proliferation, enhanced survival, and inhibition of terminal differentiation, through activation of the Ras pathway. However, the mechanism by which Fli-1 activates this signal transduction pathway has yet to be identified. Down-regulation of the Src homology 2 (SH2) domain-containing inositol-5-phosphatase-1 (SHIP-1) is associated with erythropoietin-stimulated erythroleukemic cells and correlates with increased proliferation of transformed cells. In this study, we have shown that F-MuLV-infected SHIP-1 knockout mice display accelerated erythroleukemia progression. In addition, RNA interference (RNAi)-mediated suppression of SHIP-1 in erythroleukemia cells activates the phosphatidylinositol 3-kinase (PI 3-K) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways, blocks erythroid differentiation, accelerates erythropoietin-induced proliferation, and leads to PI 3-K-dependent Fli-1 up-regulation. Chromatin immunoprecipitation and luciferase assays confirmed that Fli-1 binds directly to an Ets DNA binding site within the SHIP-1 promoter and suppresses SHIP-1 transcription. These data provide evidence to suggest that SHIP-1 is a direct Fli-1 target, SHIP-1 and Fli-1 regulate each other in a negative feedback loop, and the suppression of SHIP-1 by Fli-1 plays an important role in the transformation of erythroid progenitors by F-MuLV.

Role of phosphatidylinositol 3-kinase in friend spleen focus-forming virus-induced erythroid disease.

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin (Epo), because of interaction of the viral envelope protein with the erythropoietin receptor and a short form of the receptor tyrosine kinase Stk (sf-Stk), leading to constitutive activation of several signal transduction pathways. Our previous in vitro studies showed that phosphatidylinositol 3-kinase (PI3-kinase) is activated in SFFV-infected cells and is important in mediating the biological effects of the virus. To determine the role of PI3-kinase in SFFV-induced disease, mice deficient in the p85alpha regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85alpha status determined the extent of erythroid hyperplasia induced by the sf-Stk-dependent viruses SFFV-P (polycythemia-inducing strain of SFFV) and SFFV-A (anemia-inducing strain of SFFV) but not by the sf-Stk-independent SFFV variant BB6. Our data also indicate that p85alpha status determines the response of mice to stress erythropoiesis, consistent with a previous report showing that SFFV uses a stress erythropoiesis pathway to induce erythroleukemia. We further showed that sf-Stk interacts with p85alpha and that this interaction depends upon sf-Stk kinase activity and tyrosine 436 in the multifunctional docking site. Pharmacological inhibition of PI3-kinase blocked proliferation of primary erythroleukemia cells from SFFV-infected mice and the erythroleukemia cell lines derived from them. These results indicate that p85alpha may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia.

Gene profiling of the erythro- and megakaryoblastic leukaemias induced by the Graffi murine retrovirus.

BACKGROUND: Acute erythro- and megakaryoblastic leukaemias are associated with very poor prognoses and the mechanism of blastic transformation is insufficiently elucidated. The murine Graffi leukaemia retrovirus induces erythro- and megakaryoblastic leukaemias when inoculated into NFS mice and represents a good model to study these leukaemias. METHODS: To expand our understanding of genes specific to these leukaemias, we compared gene expression profiles, measured by microarray and RT-PCR, of all leukaemia types induced by this virus. RESULTS: The transcriptome level changes, present between the different leukaemias, led to the identification of specific cancerous signatures. We reported numerous genes that may be potential oncogenes, may have a function related to erythropoiesis or megakaryopoiesis or have a poorly elucidated physiological role. The expression pattern of these genes has been further tested by RT-PCR in different samples, in a Friend erythroleukaemic model and in human leukaemic cell lines.We also screened the megakaryoblastic leukaemias for viral integrations and identified genes targeted by these integrations and potentially implicated in the onset of the disease. CONCLUSIONS: Taken as a whole, the data obtained from this global gene profiling experiment have provided a detailed characterization of Graffi virus induced erythro- and megakaryoblastic leukaemias with many genes reported specific to the transcriptome of these leukaemias for the first time.

Establishment of mouse erythroleukemia cell lines expressing complete Influenza C virus CM2 protein or chimeric protein consisting of CM2 and Influenza A virus M2.

The role of the Influenza C virus (ICV) CM2 protein in virus replication as well as its precise function as an ion channel remains to be elucidated. For this purpose, we established a CM2-expressing mouse erythroleukemia (MEL) cell line and determined the biochemical characteristics of the expressed CM2. The features of the expressed CM2 were similar to those of the viral CM2 synthesized in ICV-infected cells. Furthermore, we established MEL cell line expressing a chimeric protein consisting of characteristic regions of CM2 and Influenza A virus (IAV) M2 protein that could be helpful in elucidation of the specific ion conductance properties.

Multi-stage Friend murine erythroleukemia: molecular insights into oncogenic cooperation.

The Friend virus SFFV (Spleen Focus Forming Virus) provokes an acute erythroblastosis in susceptible strains of mice that progresses to overt erythroleukemia by a multi-step process. For virologists, the Friend virus-induced disease has provided deep insights into the host mechanisms influencing susceptibility to retroviral infection and viremia. These insights have contributed to the understanding of HIV and other human retroviral infections. For cell biologists and oncologists, this leukemia has been a powerful experimental model to identify critical oncogenes involved in a multi-stage process, to understand the contribution of host genes to cancer development, and to investigate the mechanisms leading to cell growth autonomy. This model also provided an example of oncogenic reversion since Friend tumor cells can reinitiate their erythroid differentiation program when exposed in vitro to some chemical inducers. This review highlights recent findings demonstrating that the leukemic progression depends on the cooperation of at least two oncogenic events, one interfering with differentiation and one conferring a proliferative advantage. The Friend model of leukemia progression recapitulates the two phases of human acute myeloid leukemia (AML). Coupling of insights from studies on the Friend erythroleukemia with knowledge on AML might allow a better understanding of the molecular mechanisms involved in the evolution of leukemia in mice and men.

APRIL facilitates viral-induced erythroleukemia but is dispensable for T cell immunity and lymphomagenesis.

The TNF family member, a proliferation-inducing ligand (APRIL), has been suggested to act as a costimulatory molecule in T cell responses. However, studies addressing this role in vivo are largely lacking. Here, we evaluated the effects of APRIL on physiological T cell responses in vivo. Although receptors for APRIL are expressed on a subset of T cells, neither TCR transgenic (Tg) T cell responses nor endogenous TCR responses were affected by Tg APRIL expression in vivo. Moreover, APRIL did not significantly enhance the induction of T cell lymphomas upon Moloney murine leukemia virus (MLV) infection. This clearly contrasts current belief and indicates that APRIL does not serve a major role in T cell immunity or lymphomagenesis. However, we did observe a strong increase in erythroleukemia formation after MLV inoculation of APRIL Tg mice. Strikingly, this erythroleukemia-facilitating property of APRIL was confirmed using the erythroleukemogenic Friend-MLV. Erythroleukemia in APRIL Tg mice was characterized by low hematocrits and grossly enlarged spleens with an increased percentage of erythroid precursors. Altogether, these results unveil new proerythroleukemogenic properties of APRIL.

Friend virus utilizes the BMP4-dependent stress erythropoiesis pathway to induce erythroleukemia.

More than 50 years of genetic analysis has identified a number of host genes that are required for the expansion of infected cells during the progression of Friend-virus-induced erythroleukemia. In this report, we show that Friend virus induces the bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis pathway in the spleen, which rapidly amplifies target cells, propagating their infection and resulting in acute splenomegaly. This mechanism mimics the response to acute anemia, in which BMP4 expressed in the spleen drives the expansion of a specialized population of stress erythroid progenitors. Previously we demonstrated that these progenitors, termed stress BFU-E, are targets for Friend virus in the spleen (A. Subramanian, H. E. Teal, P. H. Correll, and R. F. Paulson, J. Virol. 79:14586-14594, 2005). Here, we extend those findings by showing that Friend virus infects two distinct populations of bone marrow cells. One population, when infected, differentiates into mature erythrocytes in an Epo-independent manner, while a second population migrates to the spleen after infection, where it induces BMP4 expression and acts as a reservoir of virus. The activation of the stress erythropoiesis pathway in the spleen by Friend virus results in the rapid expansion of stress BFU-E, providing abundant target cells for viral infection. These observations suggest a novel mechanism by which a virus induces a stress response pathway that amplifies target cells for the virus, leading to acute expansion of infected cells.

Suppression of acute anti-friend virus CD8+ T-cell responses by coinfection with lactate dehydrogenase-elevating virus.

Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8(+) T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8(+) T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8(+) T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4(+) T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies.

Inhibition of progression of erythroleukemia induced by Friend virus in BALB/c mice by natural products--berberine, curcumin and picroliv.

The infection with Friend murine leukemia virus (FMuLv) is being used as a retrovirus infection model for searching the potential anti-viral medicinal preparations or establishing new treatment strategies. In the present study we have evaluated the inhibitory effect of three non-toxic antiviral natural compounds namely berberine, curcumin or picroliv against FMuLv induced erythroleukemia in BALB/c mice. To understand the effect of these compounds in the initiation and progression of leukemia we did a series of analysis, which include hematological and biochemical parameters, histopathological evaluations of the liver and the spleen and expression analysis of selected genes such as Bcl-2, p53, p45NFE2, Raf-1, Erk-1, IFNgamma receptor and erythropoietin in spleen. The treatment with berberine, curcumin or picroliv were found to (a) elevate the life span of leukemia harboring animals by more than 60 days; (b) decreased the anemic condition which was highly prevalent in FMuLv alone treated group; (c) histopathological evaluations showed that the compounds tested here inhibited the massive leukemic cell infiltrations to sinusoidal spaces in spleen; (d) decrease the expression of Bcl-2, Raf-1, Erk-1 IFNgamma receptor and erythropoietin; (e) induce the expression of p53. Overall, our results suggest that berberine, curcumin and picroliv were able to suppress the progression of leukemia induced by FMuLv and further support their chemopreventive potential against virally induced cancers.

Enhanced natural-killer cell and erythropoietic activities in VEGF-A-overexpressing mice delay F-MuLV-induced erythroleukemia.

We have previously reported that VEGF-A, in combination with MCP-5, contributes to leukemia progression within the splenic microenvironment of mice infected with F-MuLV. To study the influence of constitutively elevated VEGF-A levels on the progression of erythroleukemia, mice heterozygous for a VEGF-A "hypermorphic" allele (Vegfhi/+) were inoculated with F-MuLV. Unexpectedly, a significant delay in erythroleukemia was observed in Vegfhi/+ mice when compared with wild-type controls. These results suggested an altered physiologic response arising from elevated VEGF-A levels that decelerated erythroleukemic progression. Characterization of hematopoiesis in Vegfhi/+ spleens showed a higher natural killer cell activity, elevated B cells, and a decrease in T-cell number. Furthermore, higher erythroid progenitors (ie, CD34+, CD36+, and Ter119+ cells) were evident in the bone marrow, spleen, and peripheral blood of Vegfhi/+ mice. The CFU-E levels were significantly elevated in Vegfhi/+ bone marrow cultures, and this elevation was blocked by a neutralizing antibody to VEGF-A receptor (VEGFR-2). Moreover, erythroleukemic mice were treated with recombinant erythropoietin and, similar to diseased Vegfhi/+ mice, showed a delay in disease progression. We propose that a compensatory erythropoietic response combined with increased natural killer (NK) cell activity account for the extended survival of erythroleukemic, Vegfhi/+ mice.

Biochemical and biophysical studies on the precursor cells of mouse erythrocytes at different stages.

The aim of this research is to study the biochemical and biophysical properties of the precursor cells of mouse erythrocytes at different stages and the molecular mechanisms of their regulation. We investigated the degree of terminal differentiation of splenic erythroblasts obtained from mice during the acute phase of disease caused by the anemia-inducing FVAstrain of Friend virus. We analyzed the transcription and protein levels of alpha-globin, beta-globin (erythroid special protein) and GATA-1 (a special erythroid transcription factor). We also have examined the Ca2+ concentration, the distribution and amount of F-actin, important cellular components such as nucleic acids, lipids, and proteins, and the adhesion of precursor cells of RBC at different stages to vascular endothelium. Our results indicated that Ca2+ concentration and the distribution and structure of F-actin changed with the development of proerythroblasts, and that the adhesion rate between the precursor cells and endothelial cells can be correlated with the expression levels of ICAM-1 and P-selectin. These alternations caused changes in biophysical properties of the cell, such as membrane fluidity and deformability.

Mutation of the Lyn tyrosine kinase delays the progression of Friend virus induced erythroleukemia without affecting susceptibility.

During the initial phase of Friend virus (FV) induced erythroleukemia, the interaction between the viral envelope glycoprotein gp55, the Erythropoietin receptor (EpoR) and the naturally occurring truncated version of the Mst1r receptor tyrosine kinase, called Sf-Stk, drives the polyclonal expansion of infected progenitors in an erythropoietin independent manner. Sf-Stk provides signals that cooperate with EpoR signals to effect expansion of erythroid progenitors. The latter phase of disease is characterized by a clonal expansion of transformed leukemic cells causing an acute erythroleukemia in mice. Signaling by Sf-Stk and EpoR mediated by gp55 renders erythroid progenitors Epo independent through the activation of the EpoR downstream pathways such as PI3K, MAPK and JAK/STAT. Previous work has shown that Src family kinases also play an important role in erythropoiesis. In particular, mutation of Src and Lyn can affect erythropoiesis. In this report we analyze the role of the Lyn tyrosine kinase in the pathogenesis of Friend virus. We demonstrate that during FV infection of primary erythroblasts, Lyn is not required for expansion of viral targets. Lyn deficient bone marrow and spleen cells are able to form Epo independent FV colonies in vitro. In vivo infection of Lyn deficient animals also results in a massive splenomegaly characteristic of the virus. However, we observe differences in the pathogenesis of Friend erythroleukemia in Lyn-/- mice. Lyn-/- mice infected with the polycythemia inducing strain of FV, FVP, do not develop polycythemia suggesting that Lyn-/- infected erythroblasts have a defect in terminal differentiation. Furthermore, the expansion of transformed cells in the spleen is reduced in Lyn-/- mice. Our data show that Lyn signals are not required for susceptibility to Friend erythroleukemia, but Lyn plays a role in later events, the terminal differentiation of infected cells and the expansion of transformed cells.