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The nitric oxide (NO) prodrug JS-K, a promising anti-cancer agent, consists of a diazeniumdiolate group necessary for the release of NO as well as an arylating ring. In this study, we research the mechanism by which JS-K kills a murine erythroleukemia cell line and determine the roles of NO and arylation in the process. Our studies indicate that JS-K inhibits the PI 3-kinase/Akt and MAP kinase pathways. This correlates with the activation of the tumor suppressor FoxO3a and increased expression of various caspases, leading to apoptosis. The arylating capability of JS-K appears to be sufficient for inducing these biological effects. Overall, these data suggest that JS-K kills tumor cells by arylating and inactivating signaling molecules that block the activation of a tumor suppressor.

Lack of suitable mouse models for central nervous system (CNS)-associated leukemias has hindered mechanism-guided development of therapeutics. By transplanting retrovirus-transformed mouse erythroleukemia cells into syngeneic mice, we developed a new animal model of meningeal leukemia associated with rapid paralysis. Necropsy revealed massive proliferation of the leukemic cells in the bone marrow (BM) followed by pathological angiogenesis and invasion of the leukemic cells into the meninges of the CNS. Further analysis demonstrated that the erythroleukemia cells secreted high levels of VEGF and preferentially adhered in vitro to fibronectin. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the BM and their invasion of the CNS as well as pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias.

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, because of the interaction among the viral envelope protein, the erythropoietin receptor, and a short form of the receptor tyrosine kinase Stk (sf-Stk). This leads to constitutive activation of several signal transduction pathways. Our previous studies showed that sf-Stk interacts with SFFV gp55, forming disulfide-linked complexes. This covalent interaction, along with other noncovalent interactions with SFFV-gp55, results in constitutive tyrosine phosphorylation of sf-Stk and rodent fibroblast transformation. Here, we determined the precise amino acid region within sf-Stk that contributes to fibroblast transformation by the polycythemia-inducing (SFFV-P) and the anemia-inducing (SFFV-A) strains of SFFV. Sf-Stk deletion mutants showed different transforming abilities in fibroblasts infected with SFFV-P and SFFV-A, although the N-terminal extracellular domain of sf-Stk was essential for fibroblast transformation by both viruses. Point mutations of sf-Stk indicated that cysteine 19 was critical for fibroblast transformation by SFFV-P, although all four cysteines (8, 19, 37 and 42) appeared to be important for fibroblast transformation by both SFFV-P and SFFV-A. Mutation of sf-Stk cysteine 19 abolished its ability to form dimers with SFFV-P and SFFV-A gp55. These results suggest that the interaction between sf-Stk and the envelope proteins of the polycythemia- and anemia-inducing variants of SFFV is architecturally different.

The cell lines of the NCI-60 panel represent different cancer types and have been widely utilized for drug screening and molecular target identification. Screening these cell lines for envelope proteins or gene sequences related to xenotropic murine leukemia viruses (X-MLVs) revealed that one cell line, EKVX, was a candidate for production of an infectious gammaretrovirus. The presence of a retrovirus infectious to human cells was confirmed by the cell-free transmission of infection to the human prostate cancer cell line LNCaP. Amplification and sequencing of additional proviral sequences from EKVX confirmed a high degree of similarity to X-MLV. The cell line EKVX was established following passage of the original tumor cells through nude mice, providing a possible source of the X-MLV found in the EKVX cells.

Hematological malignancies in humans typically involve two types of genetic changes: those that promote hematopoietic cell proliferation and survival (often the result of activation of tyrosine kinases) and those that impair hematopoietic cell differentiation (often the result of changes in transcription factors). The multi-stage erythroleukemia induced in mice by Friend spleen focus-forming virus (SFFV) is an excellent animal model for studying the molecular basis for both of these changes. Significant progress has been made in understanding the molecular basis for the multi-stage erythroleukemia induced by Friend SFFV. In the first stage of leukemia, the envelope protein encoded by SFFV interacts with and activates the erythropoietin (Epo) receptor and the receptor tyrosine kinase sf-Stk in erythroid cells, causing their Epo-independent proliferation, differentiation and survival. In the second stage, SFFV integration into the Sfpi1 locus activates the myeloid transcription factor PU.1, blocking erythroid cell differentiation, and in conjunction with the loss of p53 tumor suppressor activity, results in the outgrowth of malignant cells. In this review, we discuss the current level of understanding of how SFFV alters the growth and differentiation of erythroid cells and results in the development of erythroleukemia. Our knowledge of how SFFV causes erythroleukemia in mice may give us clues as to how the highly related human retrovirus XMRV causes malignancies in humans.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic retrovirus that has undergone genetic changes from its nonneuropathogenic parent, Friend MuLV, that allow it to efficiently infect rat brain capillary endothelial cells (BCEC). To clarify the mechanism by which PVC-211 MuLV expression in BCEC induces neurological disease, we examined virus-infected rats at various times during neurological disease progression for vascular and inflammatory changes. As early as 2 weeks after virus infection and before any marked appearance of spongiform neurodegeneration, we detected vessel leakage and an increase in size and number of vessels in the areas of the brain that eventually become diseased. Consistent with these findings, the amount of vascular endothelial growth factor (VEGF) increased in the brain as early as 1 to 2 weeks postinfection. Also detected at this early disease stage was an increased level of macrophage inflammatory protein 1α (MIP-1α), a cytokine involved in recruitment of microglia to the brain. This was followed at 3 weeks postinfection by a marked accumulation of activated microglia in the spongiform areas of the brain accompanied by an increase in tissue plasminogen activator, a product of microglia implicated in neurodegeneration. Pathological observations at the end stage of the disease included loss of neurons, decreased myelination, and mild muscle atrophy. Treatment of PVC-211 MuLV-infected rats with clodronate-containing liposomes, which specifically kill microglia, significantly blocked neurodegeneration. Together, these results suggest that PVC-211 MuLV infection of BCEC results in the production of VEGF and MIP-1α, leading to the vascular changes and microglial activation necessary to cause neurodegeneration.

Hematopoietic malignancies are frequently associated with DNA hypomethylation but the molecular mechanisms involved in tumor formation remain poorly understood. Here we report that mice lacking Lsh develop leukemia associated with DNA hypomethylation and oncogene activation. Lsh is a member of the SNF2 chromatin remodeling family and is required for de novo methylation of genomic DNA. Mice that received Lsh deficient hematopoietic progenitors showed severe impairment of hematopoiesis, suggesting that Lsh is necessary for normal hematopoiesis. A subset of mice developed erythroleukemia, a tumor that does not spontaneously occur in mice. Tumor tissues were CpG hypomethylated and showed a modest elevation of the transcription factor PU.1, an oncogene that is crucial for Friend virus induced erythroleukemia. Analysis of Lsh(-/-) hematopoietic progenitors revealed widespread DNA hypomethylation at repetitive sequences and hypomethylation at specific retroviral elements within the PU.1 gene. Wild type cells showed Lsh and Dnmt3b binding at the retroviral elements located within the PU.1 gene. On the other hand, Lsh deficient cells had no detectable Dnmt3b association suggesting that Lsh is necessary for recruitment of Dnmt3b to its target. Furthermore, Lsh(-/-) hematopoietic precursors showed impaired suppression of retroviral elements in the PU.1 gene, an increase of PU.1 transcripts and protein levels. Thus DNA hypomethylation caused by Lsh depletion is linked to transcriptional upregulation of retroviral elements and oncogenes such as PU.1 which in turn may promote the development of erythroleukemia in mice.

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope protein, gp55, which interacts with the erythropoietin (Epo) receptor complex, causing proliferation and differentiation of erythroid cells in the absence of Epo. Susceptibility to SFFV-induced erythroleukemia is conferred by the Fv-2 gene, which encodes a short form of the receptor tyrosine kinase Stk/Ron (sf-Stk) only in susceptible strains of mice. We recently demonstrated that sf-Stk becomes activated by forming a strong interaction with SFFV gp55. To examine the biological consequences of activated sf-Stk on erythroid cell growth, we prepared retroviral vectors which express sf-Stk, either in conjunction with gp55 or alone in a constitutively activated mutant form, and tested them for their ability to induce Epo-independent erythroid colonies ex vivo and disease in mice. Our data indicate that both gp55-activated sf-Stk and the constitutively activated mutant of sf-Stk induce erythroid cells from Fv-2 -susceptible and Fv-2 -resistant (sf-Stk null) mice to form Epo-independent colonies. Mutational analysis of sf-Stk indicated that a functional kinase domain and 8 of its 12 tyrosine residues are required for the induction of Epo-independent colonies. Further studies demonstrated that coexpression of SFFV gp55 with sf-Stk significantly extends the half-life of the kinase. When injected into Fv-2 -resistant mice, neither the gp55-activated sf-Stk nor the constitutively activated mutant caused erythroleukemia. Surprisingly, both Fv-2 -susceptible and -resistant mice injected with the gp55-sf-Stk vector developed clinical signs not previously associated with SFFV-induced disease. We conclude that sf-Stk, activated by either point mutation or interaction with SFFV gp55, is sufficient to induce Epo-independent erythroid colonies from both Fv-2 -susceptible and -resistant mice but is unable to cause erythroleukemia in Fv-2 -resistant mice.

A number of murine leukemia viruses (MuLVs) have been shown to induce diseases of the central nervous system (CNS) that are characterized by progressive loss of neuronal function (35, 39). The major cell types within the CNS that are prominently infected with the MuLVs are glial and endothelial cells, with neurons being infrequently infected. The most commonly observed pathological changes are gliosis, neuronal loss, and demyelination. The mechanism(s) by which the MuLVs induce neurological diseases remains to be elucidated. PVC-211 MuLV is a neuropathogenic variant of the leukemia-inducing Friend MuLV (F-MuLV) (21). Infection of susceptible rats with PVC-211 MuLV causes a rapidly progressive neurodegenerative disease characterized by tremor, spasticity, ataxia, and hind limb paralysis. Neuropathological changes include widespread perivascular gliosis, neuropil vacuolation without inflammation, and neuronal degeneration in the brain stem, cerebellum, and spinal cord (19, 28). The primary target of PVC-211 MuLV infection in the CNS is the brain capillary endothelial cell (BCEC), which is resistant to F-MuLV infection (19). The determinant of the BCEC tropism of PVC-211 MuLV was mapped to two amino acids (G116 and K129) which lie within the putative receptor binding domain of the envelope surface glycoprotein (SU) (30). Within the CNS, reactive astrocytes and degenerating neurons showed no evidence of virus infection (19). BCEC tropism of the virus has been shown to be necessary for neuropathogenesis (29), suggesting that CNS injury is indirect and that molecular events in virus-infected BCECs play a very important role in neurological disease induction. Nitric oxide (NO) is an important messenger and effector molecule involved in a number of biological functions (31). NO is synthesized from l-arginine by three isoforms of NO synthases (NOS). Endothelial cell NOS (eNOS) and neuronal NOS are constitutively expressed, and their activities are regulated by Ca2+. In contrast, inducible NOS (iNOS) is inducible and Ca2+ independent (13). In the CNS, NO may play important roles in neurotransmitter release, neurotransmitter reuptake, neurodevelopment, synaptic plasticity, and regulation of gene expression, although excessive production of NO can lead to neurotoxicity (9, 27). iNOS is an attractive candidate for mediating NO-associated neurotoxicities, because long bursts of large amounts of NO are produced by iNOS (7, 9, 32). Indeed, elevated iNOS expression has been demonstrated in such human neurological diseases as Alzheimer's disease (24) and Parkinson's disease (26). Recently, the spongiform vacuolation observed in PVC-211 MuLV-infected brains was reported to be associated with oxidative damage as detected by increased immunoreactivity for 3-nitrotyrosine (NTyr) in infected brains (43). NTyr is widely used as an indicator of NO formation, because nitration of tyrosine is mediated by reactive nitrogen species derived from NO (2, 11, 15). Elevated expression of NTyr has also been reported in human neurodegenerative diseases such as familial amyotrophic lateral sclerosis (41), Alzheimer's disease (17), Parkinson's disease (12), and human immunodeficiency virus type 1 dementia complex (5). In this study, we examined expression of iNOS and elevated expression of NTyr in PVC-211 MuLV-infected BCECs to evaluate the contribution of NO produced by infected BCECs to the neuropathogenicity induced by PVC-211 MuLV infection.

A novel protein kinase, polyploidy-associated protein kinase (PAPK), was isolated using a subtraction cDNA library approach from a mouse erythroleukemia cell line that had been induced to polyploidy after serum withdrawal. PAPK shares homology with members of the Ste20/germinal center kinase family of protein kinases and is ubiquitously expressed as two spliced forms, PAPK-A and PAPK-B, that encode for proteins of 418 and 189 amino acids, respectively. The expression of endogenous PAPK-A protein increased after growth factor withdrawal in murine hematopoietic and fibroblast cells. When tested in an in vitro kinase assay, PAPK-A was activated in response to the stress-inducing agent hydrogen peroxide and slightly by fetal calf serum. Biochemical characterization of the PAPK-A-initiated pathway revealed that this novel kinase does not affect MAP kinase activity but can stimulate both c-Jun N-terminal kinase 1 (JNK1) and ERK6/p38 gamma. The kinase activity of PAPK appears to be required for the activation of ERK6/p38 gamma but not JNK1. When an inducible construct of PAPK-A was expressed in stably transfected NIH3T3 cells, the cells exhibited distinct cytoskeletal changes and became resistant to apoptotic cell death induced by serum withdrawal, effects of PAPK that require its kinase activity. These data suggest that PAPK is a new member of the Ste20/germinal center kinase family that modulates cytoskeletal organization and cell survival.

Entry of ecotropic murine leukemia virus (MuLV) into host cells is initiated by interaction between the receptor-binding domain of the viral SU protein and the third extracellular domain (TED) of the receptor, cationic amino acid transporter 1 (CAT1). To study the molecular basis for the retrovirus-receptor interaction, mouse CAT1 (mCAT1) was expressed in human 293 cells as a fusion protein with jellyfish green fluorescent protein (GFP). Easily detected by fluorescence microscopy and immunoblot analysis with anti-GFP antibodies, the mCAT1-GFP fusion protein was expressed in an N-glycosylated form on the cell surface and in the Golgi apparatus, retaining the ecotropic receptor function. The system was applied to compare Friend MuLV (F-MuLV) and its neuropathogenic variant, PVC-211 MuLV, which exhibits a unique cellular tropism and host range, for the ability to use various CAT family members as a receptor. The results indicated that F-MuLV and PVC-211 MuLV could infect the cells expressing wild-type mCAT1 at comparable efficiencies and that rat CAT3, but not mCAT2, conferred a low but detectable level of susceptibility to F-MuLV and PVC-211 MuLV. The data also suggested that CAT proteins might be expressed in an oligomeric form. Further application of the system developed in this study may provide useful insights into the entry mechanism of ecotropic MuLV.

The proliferation and differentiation of erythroid cells is a highly regulated process that is controlled primarily at the level of interaction of erythropoietin (Epo) with its specific cell surface receptor (EpoR). However, this process is deregulated in mice infected with the Friend spleen focus-forming virus (SFFV). Unlike normal erythroid cells, erythroid cells from SFFV-infected mice are able to proliferate and differentiate in the absence of Epo, resulting in erythroid hyperplasia and leukemia. Over the past 20 years, studies have been carried out to identify the viral genes responsible for the pathogenicity of SFFV and to understand how expression of these genes leads to the deregulation of erythropoiesis in infected animals. The studies have revealed that SFFV encodes a unique envelope glycoprotein which interacts specifically with the EpoR at the cell surface, resulting in activation of the receptor and subsequent activation of erythroid signal transduction pathways. This leads to the proliferation and differentiation of erythroid precursor cells in the absence of Epo. Although the precise mechanism by which the viral protein activates the EpoR is not yet known, it has been proposed that it causes dimerization of the receptor, resulting in constitutive activation of Epo signal transduction pathways. While interaction of the SFFV envelope glycoprotein with the EpoR leads to Epo-independent erythroid hyperplasia, this is not sufficient to transform these cells. Transformation requires the viral activation of the cellular gene Sfpi-1, whose product is thought to block erythroid cell differentiation. By understanding how SFFV can deregulate erythropoiesis, we may gain insights into the causes and treatment of related diseases in man.

PVC-441 murine leukemia virus (MuLV) is a member of the PVC group of Friend MuLV (F-MuLV)-derived neuropathogenic retroviruses. In order to determine the molecular basis for the difference in neuropathogenicity between PVC-441 and the previously characterized PVC-211 MuLVs, the entire nucleotide sequence of PVC-441 MuLV was determined and compared with those of PVC-211 and F-MuLV. The results suggest that PVC-441 and PVC-211 MuLVs were formed as a result of random mutations of F-MuLV and developed differently. The distinct pathogenicities of PVC-441 and PVC-211 MuLVs were maintained in the viruses regenerated from their molecular clones, and the sequences responsible for the pathological differences observed can be localized to the env gene. The amino acid sequence of PVC-441 deduced from its nucleotide sequence revealed a number of differences from PVC-211, the most striking of which was a difference at position 129 of the SU proteins in the two viruses. Host range studies with a brain capillary endothelial cell line (RTEC-6) and Chinese hamster ovary cells (CHO-K1) revealed that PVC-441, like PVC-211, could infect these cells but its efficiency of infection was lower than that of PVC-211. These results may account for the difference in neuropathogenicity between PVC-441 and PVC-211.

PVC-211 murine leukemia virus (MuLV) causes neurodegenerative disease following inoculation of neonatal, but not adult, mice and rats. It was previously shown that tropism for brain capillary endothelial cells (CEC) was a determinant of the viral neuropathogenicity. In this study, we demonstrate that host age-dependent replication of PVC-211 MuLV in vivo occurs in CEC in the brain as well as in other organs, such as the liver, kidney, and heart. In contrast, primary explant cultures of CEC derived from brains and livers of adult and neonatal rats could be infected by PVC-211 MuLV, suggesting that the age-dependent susceptibility was abrogated in vitro. Although CEC were generally less susceptible to MuLV-mediated gene transduction than fibroblasts, treatment of CEC with 2-deoxyglucose followed by inoculation of a PVC-211 MuLV-pseudotyped vector in the absence of heparin improved the transduction efficiency. These observations support the possibility that PVC-211 MuLV may be useful for establishing models of CEC gene transduction.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) that causes a rapidly progressive neurodegenerative disease in susceptible rodents. PVC-211 MuLV, but not the parental F-MuLV, can infect rat brain capillary endothelial cells (BCEC) efficiently, and the major determinant for BCEC tropism of PVC-211 MuLV is localized within theXbaI–BamHI fragment of the viral genome containing the 5′ half of theenvgene. To further dissect theXbaI–BamHI region for its effects on BCEC tropism, we constructed recombinant viruses between PVC-211 MuLV and F-MuLV and tested their infectivity on a cell line established from rat BCEC. Our results indicated that Glu116-to-Gly and Glu129-to-Lys substitutions in the background of the F-MuLV envelope SU protein were sufficient for conferring BCEC tropism on the virus. Interference studies of these viruses on Rat-1 fibroblastic cells showed that the structure of the SU protein encoded by theXbaI–BamHI region also has significant effects on their affinity for the rat ecotropic MuLV receptor. These results support the possibility that structural elements I and II of the SU protein are important determinants for virus–receptor interaction.

Summary The Friend spleen focus-forming virus has been a valuable tool for understanding the molecular events involved in the multiple stages of leukaemia. As summarized in Figure 3, the primary effect of SFFV, which occurs within days, is to cause a polyclonal proliferation of erythroid precursor cells that can proliferate in the absence of their normal regulator erythropoietin. This is the direct result of the unique envelope glycoprotein encoded by SFFV, which is transported to the cell surface and apparently interacts with the EpoR or another component of the multimeric EpoR complex, resulting in Download full-size image Figure 3. Multistage induction of erythroleukaemia by Friend SFFV. the constitutive activation of the Epo signal transduction pathway. Within this proliferating population of erythroid cells is a rare cell that has undergone several genetic changes due to the integration of the viral genome in specific sites in the mouse DNA. This leads to the activation of a gene encoding the PU.1 transcription factor, whose high expression in erythroid cells may be the cause of the block in differentiation that is characteristic of SFFV-transformed erythroid cells. SFFV integration can also lead to the inactivation of the p53 tumour supressor gene, giving these cells a growth advantage in the mouse. The disease induced by SFFV in mice is very similar to polycythaemia vera in humans (Golde et al, 1981) . The major clinical feature of polycythaemia vera is the continuous expansion of the number of mature red blood cells in the presence of low serum Epo levels. Also, BFU-E and CFU-E from these patients can form in the absence of Epo like the analogous cells from SFFV-infected mice (Casadevall et al, 1982) . It is possible that haematopoietic cells from individuals suffering from this disease express a protein similar to the envelope glycoprotein of SFFV that can interact with the EpoR and lead to its constitutive activation. Alternatively, these patients may contain a mutant EpoR gene that is constitutively activated like the mutant EpoR described earlier. As we understand more fully how the SFFV envelope protein constitutively activates te EpoR complex, we can begin to design therapies to counteract its action that can then be applied to treating patients with polycythaemia vera or other human diseases associated with uncontrolled erythropoiesis.

We recently reported that infection of rats with the neurodegenerative disease-causing retrovirus PVC-211 MuLV results in elevated levels of the chemokine MIP-1α followed by the accumulation of activated microglia in the brain. To investigate the importance of MIP-1α in recruitment of microglia to the brain, we treated rats with MIP-1α antibodies before and after PVC-211 MuLV infection. This caused a delay in the development of paralysis which was associated with a decrease in activated microglia without affecting virus expression. To determine the source of activated microglia, rats were splenectomized 4days after virus infection. Splenectomized rats showed a delay in disease development that was associated with decreased numbers of activated microglia without affecting virus expression. Together, these results suggest that MIP-1α is directly involved in the neurodegeneration induced in rats by PVC-211 MuLV by recruiting macrophages/microglia from the periphery into regions of the brain that eventually become diseased.

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 p85α regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85α 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 p85α 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 p85α 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 p85α may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia.

The proliferation and differentiation of cells is a highly regulated process that is controlled primarily at the level of the interaction of growth factors with their cell surface receptors. However, a variety of different retroviruses alter signal transduction pathways in infected cells, which is critical for the pathogenic process. Many studies have focused on how viral oncogenes and pathogenic genes associated with these retroviruses deregulate signal transduction pathways. Protein kinases, either encoded or activated by retroviruses, appear to play a major role. In this chapter, the effects of oncogenic retroviruses on signal transduction pathways will be reviewed for four oncogenic retroviruses: Abelson murine leukemia virus, S13 avian erythroblastosis virus, Friend spleen focus-forming virus, and Jaagsiekte sheep retrovirus. Each retrovirus uses a different mechanism to deregulate cell signaling.

Recombinant human (rhu) IL-7 was evaluated for its influence on disease progression in mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC). DBA/2 mice were injected i.v. with FVC, and then treated s.c. with rhuIL-7. IL-7 significantly prolonged survival time and decreased spleen focus-forming virus (SFFV) levels, expression of SFFV mRNA and SFFV protein production in FVC-infected mice. IL-7 did not appear to directly inactivate SFFV. Although both splenic weight and cellularity in FVC-infected mice treated with IL-7 were higher than those of normal mice, they were respectively 58% and 66% lower than those of the untreated FVC-infected mice. NK-cell activity was substantially lower in FVC-infected mice than in normal mice, while IL-7 restored NK-cell activity to normal levels. IL-6 and IFN-gamma levels were markedly reduced in FVC-infected mice compared to normal mice, but treatment of FVC-infected mice with IL-7 restored these cytokine levels. While the actual mechanisms of these effects are not yet known, the results suggest the potential therapeutic efficacy of IL-7 for certain hematopoietic and viral disorders, possibly mediated through an action on accessory cells and cytokine production.

ME26 virus is a recombinant mouse retrovirus construct homologous to the avian E26 virus. Both encode a 135-kDa gag-myb-ets fusion protein which is localized in the nucleus. We have recently shown that ME26 virus can induce erythropoietin (Epo) responsiveness in hematopoietic cells. Mice infected with ME26 virus develop a hyperplasia of Epo-dependent hematopoietic precursor cells from which permanent cell lines can be established. In vitro, ME26 virus specifically induces Epo responsiveness in the interleukin-3-dependent myeloid cell line FDC-P2 by enhancing expression of the Epo receptor (EpoR). In the present study we demonstrate that ME26 virus infection of FDC-P2 cells also results in enhanced expression of beta-globin and the erythroid-specific transcription factor GATA-1, a protein which can transactivate both the EpoR promoter and globin genes. In addition, these cells exhibit a down-regulation of c-myb expression similar to that seen in differentiating erythroid cells. To determine the molecular basis for activation of erythroid genes in ME26 virus-infected cells, we carried out transient expression assays with DNA constructs of either the EpoR promoter of the GATA-1 promoter linked to reporter genes. Our results indicate that while ME26 virus did not directly enhance expression from the EpoR promoter, both it and its avian parent, E26, transactivated the GATA-1 promoter. Furthermore, ME26 virus cooperates with the GATA-1 protein to enhance expression of the EpoR gene. We propose that the mechanism by which ME26 virus induces erythroleukemia involves transactivation of the GATA-1 gene, thus positively regulating the expression of the EpoR and leading to the proliferation of a unique population of Epo-responsive cells. By specifically inducing Epo responsiveness in hematopoietic cells via transactivation of a transcription factor, ME26 virus utilizes a novel mechanism for retrovirus pathogenesis.

Induction of erythroleukemia in mice by the replication-defective spleen focus-forming virus (SFFV) relies on the presence of a helper virus to deliver the SFFV genome to erythroid target cells. Pseudotyping studies with different ecotropic murine leukemia viruses (MuLV) have shown that SFFV pseudotyped with Akv, the endogenous ecotropic virus of AKR mice, inefficiently gives rise to virus-induced erythroid bursts (vBFU-E) in vitro and fails to cause erythroleukemia in mice when compared to SFFV pseudotyped with Friend or Moloney MuLV. In order to locate the region(s) of the Akv genome responsible for its inability to act as a helper for SFFV, six different Moloney MuLV chimeras containing Akv envelope sequence substitutions were constructed. Virions with the chimeric envelopes were used to pseudotype SFFV and the complexes were analyzed for their ability to induce vBFU-E in vitro and erythroleukemia in mice. SFFV preparations pseudotyped with three of the constructs containing chimeric envelope genes efficiently gave rise to vBFU-E as did SFFV pseudotyped with Moloney MuLV. SFFV pseudotypes generated from the other three constructs, which all share a common 304-bp region located near the center of the Akv gp70 coding region, and Akv gave rise to very few vBFU-E. However, all SFFV preparations, with the exception of SFFV pseudotyped with Akv, induced erythroleukemia in mice. The results suggest that specific sequences present in the envelope gene of Akv are responsible for the inefficiency of the virus to infect erythroid target cells for SFFV, but additional Akv sequences outside those used in this study affect the ability of the Akv/SFFV virus complex to cause erythroleukemia in mice.

ME26 virus, which was generated by inserting the coding region of the acute avian leukemia-inducing virus E26 into a murine retrovirus vector, encodes a 135-kDa gag-myb-ets fusion protein. Amphotropic murine leukemia virus pseudotypes of ME26 virus induce a high incidence of erythroleukemia 2 to 4 months after injection into newborn NFS/N mice. Spleen cells from the majority of these mice proliferate to high levels in the presence of the erythroid hormone erythropoietin (Epo) and can easily be established as permanent Epo-dependent cell lines. The cell lines contain multiple copies of ME26 viral DNA and express viral message and protein. An Epo receptor mRNA of normal size can be detected in these cells, and binding studies reveal a single class of lower-affinity Epo receptor with an affinity for Epo that is in the range of that previously reported for erythroid cells. The ME26 virus-induced Epo-dependent cell lines, however, appear more immature than previously described erythroid cell lines and more closely resemble early hematopoietic precursor cells, suggesting that the virus may be activating the Epo receptor in hematopoietic cells that do not normally express it. Consistent with this idea, we are able to infect an interleukin-3-dependent myeloid cell line, FDC-P2, with ME26 virus and convert it to Epo dependence. The ME26 virus-infected FDC-P2 cells, even before growth on Epo, showed a large increase in the amount of Epo receptor mRNA. However, no ME26 viral integrations can be detected adjacent to the Epo receptor gene, indicating that the virus is not activating the Epo receptor gene by promoter/enhancer insertion. Our results are more consistent with the hypothesis that the gag-myb-ets-encoded viral fusion protein, which is known to bind DNA, is directly or indirectly activating the expression of the Epo receptor gene in these cells.

Viral Link to Chronic Fatigue Chronic fatigue syndrome (CFS) is a complex and debilitating disorder that is often linked to immune system dysfunction but whose cause(s) remain mysterious. Lombardi et al. (p. 585 , published online 8 October; see the Perspective by Coffin and Stoye ) now present a tantalizing new lead. In blood samples from 101 patients with well-documented CFS, over two-thirds (68) contained DNA from a recently described human gammaretrovirus, xenotropic murine leukemia virus–related virus (XMRV), which possesses sequence similarity to a murine leukemia virus. Cell culture assays confirmed that XMRV derived from CFS patient plasma and from T and B lymphocytes was infectious. Although the correlation with CFS is striking, whether the virus plays a causal role in the disorder remains to be determined. Interestingly, nearly 4% of the 218 healthy donors tested were positive for XMRV, which suggests that this virus—whose pathogenic potential is unknown—may be present in a significant proportion of the general population.

Erythroid cells from mice infected with the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP), unlike normal erythroid cells, can proliferate and differentiate in apparent absence of the erythroid hormone erythropoietin (Epo). The unique envelope glycoprotein encoded by SFFV has been shown to be responsible for this biological effect. The recent isolation of an Epo-dependent erythroleukemia cell line, HCD-57, derived from a mouse infected at birth with Friend murine leukemia virus, afforded us the opportunity to study the direct effect of SFFVP on a homogeneous population of factor-dependent cells. The introduction of SFFVP in complex with various helper viruses into these Epo-dependent cells efficiently and reproducibly gave rise to lines which expressed high levels of SFFV and were factor independent. SFFV appears to be unique in its ability to abrogate the factor dependence of Epo-dependent HCD-57 cells, since infection of these cells with retroviruses carrying a variety of different oncogenes had no effect. The induction of Epo independence by SFFV does not appear to involve a classical autocrine mechanism, since there is no evidence that the factor-independent cells synthesize or secrete Epo or depend on it for their growth. However, the SFFV-infected, factor-independent cells had significantly fewer receptors available for binding Epo than their factor-dependent counterparts had, raising the possibility that the induction of factor independence by the virus may be due to the interaction of an SFFV-encoded protein with the Epo receptor.

Infection of erythroid progenitor cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia and eventually to erythroleukemia in susceptible strains of mice. The viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk), activating both and inducing Epo-independent proliferation. Recently, we discovered that coexpression of SFFV gp55 and sf-Stk is sufficient to transform NIH 3T3 and primary fibroblasts. In the current study, we demonstrate that sf-Stk and its downstream effectors are critical to this transformation. Unlike SFFV-derived erythroleukemia cells, which depend on PU.1 expression for maintenance of the transformed phenotype, SFFV gp55-sf-Stk-transformed fibroblasts are negative for PU.1. Underscoring the importance of sf-Stk to fibroblast transformation, knockdown of sf-Stk abolished the ability of these cells to form anchorage-independent colonies. Like SFFV-infected erythroid cells, SFFV gp55-sf-Stk-transformed fibroblasts express high levels of phosphorylated MEK, ERK, phosphatidylinositol 3-kinase (PI3K), Gab1/2, Akt, Jun kinase (JNK), and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 mitogen-activated protein kinase (MAPK) stress response is suppressed in the transformed fibroblasts. Inhibition of either JNK or the PI3K pathway decreases both monolayer proliferation and anchorage-independent growth of the transformed fibroblasts as does the putative kinase inhibitor luteolin, but inhibition of p38 MAPK has no effect. Our results indicate that sf-Stk is a molecular endpoint of transformation that could be targeted directly or with agents against its downstream effectors.

Infection of mice with Friend spleen focus-forming virus (SFFV) results in a multistage erythroleukemia. In the first stage, the SFFV envelope glycoprotein interacts with the erythropoietin receptor and a short form of the receptor tyrosine kinase sf-Stk, resulting in constitutive activation of signal transducing molecules and the development of erythropoietin (Epo)-independent erythroid hyperplasia and polycythemia. The second stage results from the outgrowth of a rare virus-infected erythroid cell that expresses nonphysiological levels of the myeloid transcription factor PU.1. These cells exhibit a differentiation block and can be grown as murine erythroleukemia (MEL) cell lines. In this study, we examined SFFV MEL cells to determine whether their transformed phenotype was associated with a block in the activation of any Epo signal-transducing molecules. Our studies indicate that Epo- or SFFV-induced activation of STAT1/3 DNA binding activity is blocked in SFFV MEL cells. The block is at the level of tyrosine phosphorylation of STAT1, although Jak2 phosphorylation is not blocked in these cells. In contrast to Epo, alpha interferon can induce STAT1 phosphorylation and DNA binding in SFFV MEL cells. The SFFV-transformed cells were shown to express elevated levels of the hematopoietic phosphatase SHP-1, and treatment of the cells with a phosphatase inhibitor restored STAT1 tyrosine phosphorylation. MEL cells derived from Friend murine leukemia virus (MuLV) or ME26 MuLV-infected mice, which do not express PU.1, express lower levels of SHP-1 and are not blocked in STAT1/3 DNA-binding activity. Our studies suggest that SFFV-infected erythroid cells become transformed when differentiation signals activated by STAT1/3 are blocked due to high SHP-1 levels induced by inappropriate expression of the PU.1 protein.

Friend spleen focus-forming virus (SFFV) causes rapid erythroleukemia 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 constitutive activation of Epo signal transduction pathways. Although SFFV infects many cell types, deregulation of cell growth occurs only when SFFV infects erythroid cells, suggesting that these cells express unique proteins that the virus requires to mediate its biological effects. Not only do erythroid cells express the Epo receptor (EpoR), but those from mice susceptible to SFFV-induced erythroleukemia also express a short form of the receptor tyrosine kinase Stk (sf-Stk). In erythroid cells, SFFV gp55 interacts with the EpoR complex and sf-Stk, leading to activation of the kinase and constitutive activation of signal transducing molecules. In this study, we demonstrate that SFFV gp55 can also deregulate the growth of nonerythroid cells when it is coexpressed with sf-Stk. Expression of SFFV gp55 in rodent fibroblasts engineered to express sf-Stk induced their transformation, as demonstrated by focus formation and anchorage-independent growth in vitro . This transformation by SFFV gp55 requires the kinase activity of sf-Stk and the presence of its extracellular domain but not expression of the EpoR or the tyrosine kinase Jak2, which is required for activation of signal transduction pathways through the EpoR. Thus, expression of SFFV gp55 in nonerythroid cells coexpressing sf-Stk results in their uncontrolled growth, demonstrating a previously unrecognized mechanism for retrovirus transformation of rodent fibroblasts and providing insight into SFFV-induced disease.

Members of the mitogen-activated protein kinase (MAPK) family, including Jun amino-terminal kinase (JNK) and extracellular signal-related kinase (ERK), play an important role in the proliferation of erythroid cells in response to erythropoietin (Epo). Erythroid cells infected with the Friend spleen focus-forming virus (SFFV) proliferate in the absence of Epo and show constitutive activation of Epo signal transduction pathways. We previously demonstrated that the ERK pathway was constitutively activated in Friend SFFV-infected erythroid cells, and in this study JNK is also shown to be constitutively activated. Pharmacological inhibitors of both the ERK and JNK pathways stopped the proliferation of primary erythroleukemic cells from Friend SFFV-infected mice, with little induction of apoptosis, and furthermore blocked their ability to form Epo-independent colonies. However, only the JNK inhibitor blocked the proliferation of erythroleukemia cell lines derived from these mice. The JNK inhibitor caused significant apoptosis in these cell lines as well as an increase in the fraction of cells in G 2 /M and undergoing endoreduplication. In contrast, the growth of erythroleukemia cell lines derived from Friend murine leukemia virus (MuLV)-infected mice was inhibited by both the MEK and JNK inhibitors. JNK is important for AP1 activity, and we found that JNK inhibitor treatment reduced AP1 DNA-binding activity in primary erythroleukemic splenocytes from Friend SFFV-infected mice and in erythroleukemia cell lines from Friend MuLV-infected mice but did not alter AP1 DNA binding in erythroleukemia cell lines from Friend SFFV-infected mice. These data suggest that JNK plays an important role in cell proliferation and/or the survival of erythroleukemia cells.

We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.

Previous studies from our laboratory demonstrated that PVC-211 murine leukemia virus (MuLV), a neuropathogenic variant of Friend MuLV (F-MuLV), had undergone genetic changes which allowed it to efficiently infect rat brain capillary endothelial cells (BCEC) in vivo and in vitro. Two amino acid changes from F-MuLV in the putative receptor binding domain (RBD) of the envelope surface protein of PVC-211 MuLV (Glu-116 to Gly and Glu-129 to Lys) were shown to be sufficient for conferring BCEC tropism on PVC-211 MuLV. Recent examination of the unique RBD of PVC-211 MuLV revealed that the substitution of Lys for Glu at position 129 created a new heparin-binding domain that overlapped a heparin-binding domain common to ecotropic MuLVs. In this study we used heparin-Sepharose columns to demonstrate that PVC-211 MuLV, but not F-MuLV, can bind efficiently to heparin and that one or both of the amino acids in the RBD of PVC-211 MuLV that are associated with BCEC tropism are responsible. We further showed that heparin can enhance or inhibit MuLV infection and that the mode of action is dependent on heparin concentration, sulfation of heparin, and the affinity of the virus for heparin. Our results suggest that the amino acid changes that occurred in the envelope surface protein of PVC-211 MuLV may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor and efficiently infect these cells.

The Friend spleen focus-forming virus (SFFV) causes an acute erythroleukemia in susceptible strains of mice (for a review, see reference 37). SFFV encodes a unique envelope glycoprotein, gp55, that associates specifically with the erythropoietin receptor (EpoR) at the cell surface (4, 10, 21, 45), allowing erythroid cells to proliferate in the absence of erythropoietin (Epo), the normal regulator of erythropoiesis. Epo stimulation of the EpoR activates a number of signal transduction pathways, including the Jak-Stat, the Ras/Raf-1/mitogen-activated protein kinase (MAPK), and the phosphatidylinositol 3-kinase (PI3-kinase) pathways (for a review, see reference 46). Using the Epo-dependent erythroleukemia cell line HCD-57, we have previously demonstrated that infection with SFFV, which abrogates the Epo dependence of these cells (36), constitutively activates Stat DNA-binding activity (30); Ras (27); Raf-1, MEK, and MAPK (26); PI 3-kinase and Akt kinase (29); and protein kinase C (27). Although interaction of the SFFV envelope glycoprotein with the EpoR complex is essential for inducing the biological effects of the virus, other factors must also be involved, since not all strains of mice are susceptible to SFFV-induced erythroleukemia. A number of polymorphic mouse genes have been identified that affect susceptibility to Friend SFFV-induced erythroleukemia. Most of these host genes interfere with viral entry or integration, or they control immune responses against the virus-infected cells (for a review, see reference 7). However, the Fv-2 gene (22), which is located on mouse chromosome 9, acts at the level of the erythroid target cell for the virus (2, 3, 9, 13, 39). Mice carrying at least one copy of the Fv-2s allele are susceptible to SFFV-induced erythroleukemia, and their erythroid cells will form Epo-independent erythroid bursts when infected in vitro with SFFV. In contrast, mice homozygous for the Fv-2r allele fail to develop SFFV-induced erythroleukemia, and SFFV infection of erythroid cells from these mice does not result in the formation of Epo-independent erythroid bursts. Thus, erythroid cells from Fv-2-resistant mice appear to lack a component necessary for mediating the biological effects of SFFV infection. Although earlier studies suggested that there were differences between Fv-2-susceptible and -resistant erythroid cells in cell cycling (42), it was unclear how this affected the ability of SFFV to alter the growth of these cells. Recently, however, it was shown that the Fv-2 gene is identical to the gene encoding the Met-related tyrosine kinase Stk/Ron (33). Mice that carry the Fv-2s allele express both a full-length Stk and a truncated form of the kinase, sf-Stk, which is expressed from an alternate promoter. sf-Stk lacks almost the entire extracellular domain but retains the transmembrane and tyrosine kinase domains. In contrast to Fv-2s mice, mice that are homozygous for the Fv-2r allele express only full-length Stk due to a 3-nucleotide deletion in the alternate promoter. Transfer of bone marrow cells expressing sf-Stk to Fv-2rr mice confers susceptibility to SFFV-induced erythroleukemia, while targeted disruption of the Stk gene in Fv-2s mice results in resistance to disease (33). Thus, expression of sf-Stk in erythroid cells appears to play a critical role in determining the biological consequences of SFFV infection in the mouse. We, therefore, carried out studies to determine if SFFV gp55 interacts with sf-Stk and whether this interaction results in the activation of this truncated kinase and the downstream activation of signal transduction pathways.

The ubiquitin-dependent proteasome-mediated (Ub-Pr) degradation pathway has been shown to regulate a large variety of substrates, including nuclear, cytosolic, and membrane proteins. In mammalian systems, polyubiquitin modification has been identified in a number of cell surface receptors for more than a decade; however, its biological significance has remained unclear until recently. For growth factor receptors with intrinsic tyrosine kinase domains, polyubiquitination is believed to trigger the internalization and subsequent degradation via the lysosomal pathway. In this study we provide the first evidence that non-tyrosine kinase-type cytokine surface receptors, IL-9R α-chain, IL-2 receptor β-chain, and erythropoietin receptor, can be polyubiquitinated and degraded by proteasomes. The Ub-Pr pathway regulates both the basal level turnover and the ligand-induced degradation of the receptors. A previously identified putative molecular chaperon, valosin-containing protein, undergoes tyrosine phosphorylation in a cytokine-dependent manner and associates with the receptor complexes following receptor engagement, suggesting that valosin-containing protein may target the ubiquitinated receptors to the proteasome for degradation.

Interaction of erythropoietin (Epo) with its cell surface receptor activates signal transduction pathways which result in the proliferation and differentiation of erythroid cells. Infection of erythroid cells with the Friend spleen focus-forming virus (SFFV) leads to the interaction of the viral envelope glycoprotein with the Epo receptor and renders these cells Epo independent. We previously reported that SFFV induces Epo independence by constitutively activating components of several Epo signal transduction pathways, including the Jak-Stat and the Raf-1/mitogen-activated protein kinase (MAPK) pathways. To further evaluate the mechanism by which SFFV activates the Raf-1/MAPK pathway, we investigated the effects of SFFV on upstream components of this pathway, and our results indicate that SFFV activates Shc and Grb2 and that this leads to Ras activation. While studies with a dominant-negative Ras indicated that Ras was required for Epo-induced proliferation of normal erythroid cells, the Epo-independent growth of SFFV-infected cells can still occur in the absence of Ras, although at reduced levels. In contrast, protein kinase C (PKC) was shown to be required for the Epo-independent proliferation of SFFV-infected cells. Further studies indicated that PKC, which is thought to be involved in the activation of both Raf-1 and MAPK, was required only for the activation of MAPK, not Raf-1, in SFFV-infected cells. Our results indicate that Ras and PKC define two distinct signals converging on MAPK in both Epo-stimulated and SFFV-infected erythroid cells and that activation of only PKC is sufficient for the Epo-independent proliferation of SFFV-infected cells.

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). In an attempt to understand how the virus causes Epo independence, we have been studying signal transduction pathways activated by Epo to determine if SFFV exerts its biological effects by constitutively activating any of these pathways in the absence of Epo. We previously demonstrated that Stat proteins, the downstream components of the Epo-induced Jak-Stat pathway, are constitutively activated in SFFV-infected cells. In this study, we demonstrate that SFFV also activates Raf-1, MEK and mitogen-activated protein (MAP) kinase, the downstream components of the Raf-1/MAP kinase pathway. This pathway was activated in cells infected with the polycythemia-inducing strain of SFFV, which induces both proliferation and differentiation of erythroid cells in the absence of Epo, as well as in cells infected with the anemia-inducing strain of the virus, which still require Epo for differentiation. Inhibition of Raf-1 by using antisense oligonucleotides led to a partial inhibition of the Epo-independent proliferation of SFFV-infected cells. Expression of the transcription factors c-Jun and JunB, but not c-Fos, was induced in SFFV-infected cells in the absence of Epo, suggesting that constitutive activation of the Raf-1/MAP kinase pathway by the virus may result in deregulation of AP-1 activity. We conclude from our studies that infection of erythroid cells with SFFV leads to the constitutive activation of signal transduction molecules in both the Jak-Stat and Raf-1/MAP kinase pathways and that both of these pathways must be activated to achieve maximum proliferation and differentiation of erythroid cells in the absence of Epo.

The signal transduction mechanism of erythropoietin (Epo), which regulates growth and differentiation of erythroid cells, is still unclear. Recent studies showing the activation by various ligands of a group of proteins called Stat (signal transducers and activators of transcription) proteins raised the possibility that such proteins may also be involved in the Epo signal transduction pathway. In this report, we show that Epo induces factors that specifically bind to the sis-inducible element and the gamma response region of the Fc gamma receptor factor I gene in the Epo-dependent mouse erythroleukemia cell line HCD-57. These factors contain phosphotyrosine and antibodies against Stat1 and Stat3 proteins reacted with them. In HCD-57 cells infected with Friend spleen focus-forming virus, which now grow in an Epo-independent manner, the DNA-binding factors were constitutively activated even in the absence of Epo. These results suggest that the factors induced by Epo contain components identical or related to known Stat proteins. It is also suggested that continuous activation of these DNA-binding factors may be responsible for the ability of spleen focus- forming virus to abrogate the Epo-dependence of HCD-57 cells and cause erythroleukemia in susceptible mice.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic, weakly leukemogenic variant of the nonneuropathogenic, highly leukemogenic Friend MuLV (F-MuLV). Chimeric viruses constructed from PVC-211 MuLV clone 3d and F-MuLV clone 57 indicate that the env gene of PVC-211 MuLV contains the determinant(s) responsible for pathological changes in the central nervous system. However, sequences within the 5' one-third (AatII-EcoRI region) of the PVC-211 MuLV genome, which include the 5' leader sequence, the gag gene, and the 5' quarter of the pol gene, are also needed in conjunction with the env gene determinant(s) to cause clinically evident neurological disease in the majority of virus-infected animals after a short latency. In the presence of the AatII-EcoRI region of the PVC-211 MuLV genome, the PVC-211 MuLV env gene sequences encoding the amino-terminal half of the SU protein, which contains the receptor-binding region of the protein, were sufficient to cause rapidly progressive neurological disease. When PVC-211 MuLV, F-MuLV, and various chimeric viruses were tested for their ability to replicate in cultured brain capillary endothelial cells (BCEC), the primary site of PVC-211 MuLV replication within the central nervous system, there was a direct correlation between the replication efficiency of a virus in BCEC in vitro and its ability to cause neurological disease in vivo. This observation indicates that the sequences in PVC-211 MuLV that render it neuropathogenic affect its replication in BCEC and suggests that rapid and efficient replication of the virus in BCEC is crucial for the pathological changes in the central nervous system that result in development of neurological disease.

PVC-211 murine leukemia virus (MuLV) is a replication-competent, ecotropic type C retrovirus that was isolated after passage of the Friend virus complex through F344 rats. Unlike viruses in the Friend virus complex, it does not cause erythroleukemia but causes a rapidly progressive hind limb paralysis when injected into newborn rats and mice. We have isolated an infectious DNA clone (clone 3d) of this virus which causes neurological disease in animals as efficiently as parental PVC-211 MuLV. The restriction map of clone 3d is very similar to that of the nonneuropathogenic, erythroleukemogenic Friend murine leukemia virus (F-MuLV), suggesting that PVC-211 MuLV is a variant of F-MuLV and that no major structural alteration was involved in its derivation. Studies with chimeric viruses between PVC-211 MuLV clone 3d and wild-type F-MuLV clone 57 indicate that at least one determinant for neuropathogenicity resides in the 2.1-kb XbaI-ClaI fragment containing the gp70 coding region of PVC-211 MuLV. Compared with nonneuropathogenic ecotropic MuLVs, the env gene of PVC-211 MuLV encodes four unique amino acids in the gp70 protein. Nucleotide sequence analysis also revealed a deletion in the U3 region of the long terminal repeat (LTR) of PVC-211 MuLV clone 3d compared with F-MuLV clone 57. In contrast to the env gene of PVC-211 MuLV, particular sequences within the U3 region of the viral LTR do not appear to be required for neuropathogenicity. However, the changes in the LTR of PVC-211 MuLV may be responsible for the failure of this virus to cause erythroleukemia, because chimeric viruses containing the U3 region of F-MuLV clone 57 were erythroleukemogenic whereas those with the U3 of PVC-211 MuLV clone 3d were not.

The binding constants of antibodies to poly(Glu52Lys33Tyr15) from several strains of high and low responding rats and their various hybrids were measured by equilibrium dialysis. High responders produced antibodies with higher binding constants than low responders. Breeding studies showed that the ability to produce high affinity antibodies is under genetic control and is linked to the major histocompatibility locus. Aggregation of the antigen increased the quantity of antibody and its binding constant in the low responders and decreased both in the high responders. Precipitin reactions using various related synthetic polypeptides indicated that the antibodies produced by the high responders were more cross-reactive than those produced by the low responders. Breeding studies showed that this ability is also inherited in a dominant manner and is linked to the major histocompatibility locus.

A high-titer, recombinant retroviral vector produced in psi 2 packaging cells has been used to introduce the murine interleukin-3 (IL-3) gene into mouse hematopoietic cells. Integration and expression of the IL-3 gene was observed in spleen foci from which could be derived factor-independent, continuously proliferating cell lines. Irradiated or genetically anemic W/Wv recipients of infected hematopoietic cells developed a myeloproliferative syndrome characterized by a marked elevation in leukocyte count, bone marrow hyperplasia, and enlargement of the liver and spleen. The syndrome reflected proliferation of one or more stem cell clones, the progeny of which were capable of repopulating secondary recipients. One animal developed the syndrome primarily by a paracrine mechanism. Endogenous IL-3 production caused amplification of hematopoietic cells but did not appear to alter the maturational or self-renewal potential of these cells.

The genome of the Lilly-Steeves strain of spleen focus-forming virus (SFFV) was molecularly cloned in the plasmid vector pBR322. Infectious SFFV could be recovered by releasing the SFFV DNA from the vector, transfecting the released DNA onto NIH 3T3 cells, and rescuing the SFFV either by superinfection with helper virus or by cotransfection with molecularly cloned infectious helper viral DNA. By using transfections with SFFV DNA still attached to the plasmid vector, infectious SFFV activity could also be recovered with either method of rescue. Studies performed with these latter types of transfections indicated that only a portion of the SFFV genome was required for biological activity. Since gp52, a marker protein for SFFV, could be detected in all cultures from which adequate titers of biologically active SFFV were recovered, the results are consistent with the hypothesis that gp52 is necessary for SFFV-induced erythroblastosis and polycythemia.

Helper-independent virus from a molecularly cloned stock of Friend murine leukemia virus (F-MuLV) induces an erythroproliferative disease characterized by splenomegaly and severe anemia in newborn mice of certain susceptible strains, such as NIH Swiss. With age, NIH Swiss mice become resistant to this F-MuLV-induced erythroleukemia. Resistance is not correlated with the replication of the input Friend MuLV but is correlated related with the replication of Friend MCF viruses in the spleens of infected mice. Treatment of adult NIH Swiss mice with X-irradiation, phenylhydrazine, or silica before injection of F-MuLV increases the replication of MCF viruses in the spleens of these mice and renders them significantly more susceptible to erythroleukemia. In contrast to mice infected with F-MuLV as newborns or as X-irradiated adults, untreated 6-week-old NIH Swiss mice develop a good humoral immune response to MCF gp70. These results suggest that the resistance of adult NIH Swiss mice to erythroleukemia induced by F-MuLV is due to the lack of replication of Friend MCF viruses in these mice because of (1) a good immune response to the virus, (2) the lack of a suitable number of susceptible target cells, or (3) both.

The structure of the envelope gene product (gp52) of the spleen focus-forming virus was analyzed and compared to that of envelope proteins (gp70 and p15E) of another pathogenic Friend virus recombinant, Friend mink cell focus-inducing virus (F-MCF). This has enabled us to confirm and extend previous nucleotide sequence data regarding the make up of specific domains of the SFFV glycoprotein. Amino-terminal 23,000-Da V8 protease fragments from gp52 of the Lilly-Steeves strain of SFFV and from gp70 of a pathogenic F-MCF isolate produced tryptic peptide fingerprints in which the mobilities of the trypsin-generated peptides were identical. The carboxyl-terminal, 21,000 Da, V8 protease fragment of gp52, however, has a unique fingerprint that contained a single highly charged trypsin-generated peptide. This peptide migrated to the same position as a peptide in F-MCF p15E, thus indicating that the p15E-related nucleotide sequences, that follow the large envelope deletion, are translated in the same reading frame as those in the standard p15E. Although R peptide determinants can be detected in F-MCF Pr15E, they could not be detected in gp52. The amino-terminal 23K domain of gp52, like that of MCF gp70, contains two oligosaccharide attachment sites. The other two attachment sites are located within the 21K carboxyl-terminal domain.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to be associated with active centers of hematopoiesis and lymphopoiesis in the developing fetus. Therefore, the effects of TGF-beta 1 on mouse hematopoiesis were studied. TGF-beta 1 is a potent inhibitor of IL-3-induced bone marrow proliferation, but it does not inhibit the proliferation induced by granulocyte/macrophage, colony-stimulating factor (CSF), granulocyte CSF, and erythropoietin (Epo). TGF-beta 1 also inhibits IL-3-induced multipotential colony formation of bone marrow cells in soft agar, which includes early erythroid differentiation, while Epo-induced terminal differentiation is unaffected. In addition, IL-3-induced granulocyte/macrophage colonies were inhibited; however, small clusters of differentiated myeloid cells were consistently seen in cultures containing IL-3 and TGF-beta 1. Thus, TGF-beta 1 selectively inhibits early hematopoietic progenitor growth and differentiation but not more mature progenitors. TGF-beta 1 is also a potent inhibitor of IL-3-dependent and -independent myelomonocytic leukemic cell growth, while the more mature erythroid and macrophage leukemias are insensitive. Therefore, TGF-beta 1 functions as a selective regulator of differentiating normal hematopoietic cells, and suppresses myeloid leukemic cell growth.