Heterologous viral promoters incorporated into the human cytomegalovirus genome are silenced during experimental latency.

Human cytomegalovirus (HCMV) lytic phase gene expression is repressed upon entry into myeloid lineage cells where the virus establishes latency. Lytic infection is not initiated because the tegument-delivered transactivator protein pp71 fails to enter the nucleus and inactivate the Daxx-mediated cellular intrinsic defense that silences the viral genome. When pp71 is expressed de novo in THP-1 monocytes, it localizes to the nucleus, inactivates the Daxx defense, and initiates lytic infection. We speculated that replacing the native viral promoter that drives pp71 expression with one that is highly and constitutively active in myeloid cells would permit pp71 de novo expression upon infection and that this newly expressed pp71 would accumulate in the nucleus, inactivate the intrinsic defense, and initiate the cascade of lytic gene expression. Surprisingly, we found that this promoter was still subject to normal silencing mechanisms in THP-1 monocytes and primary CD34(+) cells, two independent myeloid lineage cells. A second constitutively active heterologous viral promoter located in a different region of the HCMV genome was also silenced in THP-1 and CD34(+) cells. Furthermore, these two independent heterologous viral promoters inserted into three different regions of the HCMV genome in three different viral strains all required prior expression of the viral immediate early proteins for activation in fibroblasts. From this, we conclude that incorporation within the HCMV genome impacts the proclivity of heterologous viral promoters to initiate transcription. These observations have mechanistic implications for the expression of viral genes and transgenes during both lytic infection and latency.

The tyrosine kinase sf-Stk and its downstream signals are required for maintenance of friend spleen focus-forming virus-induced fibroblast transformation.

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.

Selection of an optimal promoter for gene transfer in normal B cells.

Gene transfer into normal quiescent human B cells is a challenging procedure. The present study aimed to investigate whether it is possible to increase the levels of transgene expression by using various types of promoters to drive the expression of selected genes­of­interest. To produce lentiviral particles, the present study used the 2nd generation psPAX2 packaging vector and the vesicular stomatitis virus ­expressing envelope vector pMD2.G. Subsequently, lentiviral vectors were generated containing various promoters, including cytomegalovirus (CMV), elongation factor­1 alpha (EF1α) and spleen focus­forming virus (SFFV). The present study was unable to induce satisfactory transduction efficiency in quiescent normal B cells; however, infection of normal B cells with Epstein­Barr virus resulted in increased susceptibility to lentiviral transduction. In addition, the SFFV promoter resulted in a higher level of transgene expression compared with CMV or EF1α promoters. As a proof­of concept that this approach allows for stable gene expression in normal B cells, the present study used bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins, as well as X­box binding protein­1 and binding immunoglobulin protein.

Protective influence of lactoferrin on mice infected with the polycythemia-inducing strain of Friend virus complex.

Purified iron-saturated human lactoferrin (LF) was assessed in vivo for effects on the survival rates of C57BL X DBA/2 f1 (hereafter called BD2F1) (Fv-2sr) mice and titers of spleen focus-forming viruses (SFFV) in BD2F1 and DBA/2 (Fv-2ss) mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). LF prolonged the survival rates and decreased the titers of SFFV in mice given FVC-P. Titers of SFFV, assayed 14 days after administration of FVC-P, were measured by the spleen focus-forming unit assay in secondary mouse recipients. Decreases in titers of SFFV were apparent when LF was given in vivo as a single bolus dose of 200 micrograms within 2 h of the Friend virus complex (FVC), or as a total dosage of 200 micrograms given on days 1, 2, 4, 7, 9, and 11 after FVC-P, and to a lesser degree when LF was given as a total dosage of 200 micrograms on days 3, 4, 7, 9, and 11 after FVC-P. No decreases in titers of SFFV were detected when LF was given up to 3 days before or more than 3 days after FVC-P. LF did not appear to be directly inactivating the viruses as it did not inactivate the SFFV or the Friend murine leukemia helper virus in vitro. The results suggest that the protective effect of LF in vivo is probably due to an action on cells responding to the FVC or to an action on cells which influence the cells responding to the FVC or which influence the virus. It has been shown elsewhere that LF decreases the percentage of marrow and spleen hematopoietic progenitor cells that are in DNA synthesis in vivo and this may be the means by which the protective effect of LF is mediated in mice given the FVC.

Ets and retroviruses - transduction and activation of members of the Ets oncogene family in viral oncogenesis.

Studies of retroviral-induced oncogenesis in animal systems led to the initial discovery of viral oncogenes and their cellular homologs, and provided critical insights into their role in the neoplastic process. V-ets, the founding member of the ETS oncogene family, was originally identified as part of the fusion oncogene encoded by the avian acute leukemia virus E26 and subsequent analysis of virus induced leukemias led to the initial isolation of two other members of the ETS gene family. PU.1 was identified as a target of insertional activation in the majority of tumors induced by the murine Spleen Focus Forming virus (SFFV), while fli-1 proved to be the target of Friend murine leukemia virus (F-MuLV) in F-MuLV induced erythroleukemia, as well as that of the 10A1 and Graffi viruses. The common features of the erythroid and myeloid diseases induced by these viruses provided the initial demonstration that these and other members of the ETS family play important roles in hematopoietic development as well as disease. This review provides an overview of the role of ETS genes in retrovirally induced neoplasia, their possible mechanisms of action, and how these viral studies relate to current knowledge of the functions of these genes in hematopoiesis.

Oncogene cooperativity in Friend erythroleukemia: erythropoietin receptor activation by the env gene of SFFV leads to transcriptional upregulation of PU.1, independent of SFFV proviral insertion.

Cancer is a multi-step, multi-genetic event. Whether oncogenic mutations cooperate with one another to transform cells and how is not well understood. The Friend murine retroviral erythroleukemia model involves mitogenic activation of the erythropoietin receptor (EpoR) by the virus env gene (F-gp55), aberrant over-expression of the transcription factor PU.1, and inactivating mutations in p53. In this report we demonstrate that concurrent expression of F-gp55 and PU.1 in erythroid target cells, in vivo, cooperate to accelerate erythroleukemia induction. Early in the disease, prior to the detection of clonal leukemic cells, activation of the EpoR by F-gp55, but not erythropoietin, resulted in transcriptional upregulation of PU.1 through a trans regulatory mechanism. This could occur in the absence of an integrated provirus within the PU.1 gene locus. The regulation of PU.1 transcription in established erythroleukemia cell lines differed depending upon the level of PU.1 protein present. Our results suggest that the action of F-gp55 contributes to both early and late stages of Friend erythroleukemia and that persistence of F-gp55 expression may be required not only to initiate erythroleukemia but to also maintain erythroleukemia following Friend virus infection.

Mutational analysis of the structure-function relationship of the leukemogenic membrane glycoprotein (GP55) of Friend spleen focus-forming virus (F-SFFV).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in adult mice. F-SFFV encodes an envelope protein-like membrane glycoprotein called gp55 in its defective env gene. Gp55 is responsible for the early stage of leukemogenesis by F-SFFV by specifically binding to and activating the murine erythropoietin receptor (EPO-R). Gp55 has a polytropic env sequence in its N-terminal portion. This portion probably contains the binding site for the EPO-R. In order to obtain a clue for the structure of the binding site to the EPO-R, we isolated and analyzed many spontaneous revertant F-SFFVs which derived from the non-leukemogenic mutant F-SFFV having an ecotropic env sequence instead of the polytropic env sequence in its gp55 gene.

Activation of the JAK1-STAT5 pathway by binding of the Friend virus gp55 glycoprotein to the erythropoietin receptor.

Friend spleen focus forming-virus (F-SFFV) induces acute erythroleukemia in susceptible mice. Initiation of the erythroleukemia is due to binding of the env-related glycoprotein gp55 encoded by F-SFFV to the erythropoietin receptor (EPOR). The gp55/EPOR interaction induces prolonged and growth factor independent proliferation in a factor-dependent cell line. In erythropoietin (EPO) signaling, the JAK2/STAT5 pathway was shown to be activated downstream of the EPOR to transmit the signal to the cells. To determine members of the JAK family and the STAT transcription factor family involved in the gp55/EPOR signaling, we examined tyrosine phosphorylation of JAKs and STATs in F-SFFV-infected erythroid or erythroleukemic cells. JAK1 and STAT5 were constitutively tyrosine-phosphorylated but the DNA binding activity of STAT5 was not induced without EPO stimulation in erythroblastoid cells from spleens of F-SFFV-infected mice and erythroleukemia cell lines derived from gp55-transgenic mice. These results indicate that JAK1 is involved in the gp55/EPOR signaling but STAT5 is not playing an essential role in the growth of those erythroid cells.

Constitutive activation of Stat-related DNA-binding proteins in erythroid cells by the Friend spleen focus-forming virus.

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 the erythroid hormone erythropoietin (Epo). In an attempt to understand how the virus alters the growth of erythroid cells, studies were carried out to determine if virus infection leads to the constitutive activation of the Jak-Stat pathway, one of the signal transduction pathways activated by Epo. Our data indicates that expression of SFFV in erythroid cells leads to the constitutive activation of the same Stat proteins that are transiently activated by Epo. While constitutive activation of Stat proteins by SFFV is associated with Epo-independent proliferation of splenic erythroid progenitor cells from Fv-2-sensitive mice and Epo-dependent HCD-57 cells, it is not sufficient to induce their differentiation. Although constitutive activation of the same Stat proteins is detected in erythroid cells from SFFV-infected Fv-2-resistant mice, it does not lead to their Epo-independent growth. It is also not required for transformation of erythroid cells by SFFV. Studies are in progress to identify the mechanism by which Stat proteins are phosphorylated in SFFV-infected cells in the absence of Epo. Although it has been shown that Epo activates Stat proteins through Jak2 kinase, our results suggest that the SFFV-induced Stat protein activation is Jak2-independent.

Amplified and tissue-directed expression of retroviral vectors using ping-pong techniques.

Ping-pong amplification is an efficient process by which helper-free retrovirions replicate in cocultures of cell lines that package retroviruses into distinct host-range envelopes [11]. Transfection of a retroviral vector DNA into these cocultures results in massive virus production, with potentially endless cross-infection between different types of packaging cells. Because the helper-free virus spreads efficiently throughout the coculture, it is unnecessary to use dominant selectable marker genes, and the retroviral vectors can be simplified and optimized for expressing a single gene of interest. The most efficient ping-pong vector, pSFF, derived from the Friend erythroleukemia virus, has been used for high-level expression of several genes that could not be expressed with commonly employed two-gene retroviral vectors. Contrary to previous claims, problems of vector recombination are not inherent to ping-pong methods. Indeed, the pSFF vector has not formed replication-competent recombinants as shown by stringent assays. Here we review these methods, characterize the ping-pong process using the human erythropoietin gene as a model, and describe a new vector (pSFY) designed for enhanced expression in T lymphocytes. Factors that limit tissue-specific expression are reviewed.

Detection of ecotropic replication-competent retroviruses: comparison of s(+)/l(-) and marker rescue assays.

Guidelines for testing gene therapy products for ecotropic replication-competent retrovirus (Eco-RCR) have not been delineated as they have for amphotropic viruses. To evaluate biologic assays that can detect these viruses, we compared an S(+)/L(-) assay and a marker rescue assay designed specifically for Eco-RCR detection. Moloney murine leukemia virus (Mo-MuLV) obtained from the American Type Culture Collection was used as the positive control. For marker rescue, NIH 3T3 cells were transduced with a retroviral vector expressing the neomycin phosphotransferase gene (3T3/Neo). Inoculation and passage of test material in 3T3/Neo cells for 3 weeks (amplification) and subsequent testing in the S(+)/L(-) assay or the marker rescue assay increased the level of sensitivity for virus detection greater than 10-fold compared with direct inoculation of D56 S(+)/L(-) cells. When serial dilutions of Mo-MuLV stock were evaluated, six of six cultures had detectable virus by the S(+)/L(-) and marker rescue assays at dilutions of 10(-5) and 10(-6). At the 10(-7) dilution, five of six assays had detectable virus in both assays. The ability to detect virus-infected cells was also evaluated in a modification that substituted cells for supernatant. Fifteen 3T3/Neo cultures inoculated with 10(6) 293 cells containing 100 or 10 Mo-MuLV/3T3 cells were all positive by marker rescue. For dilution with 1 virus-infected cell per 10(6) 293 cells, 10 of 15 cultures were positive. At the 0.1-cell dilution only 2 of 15 cultures were positive. If we hope to detect one infected cell in a test article, the probability of detecting virus if the assay is performed in triplicate is 96.3%. In summary, after 3 weeks of amplification the S(+)/L(-) and marker rescue assays can detect virus with similar sensitivities. We prefer the marker rescue assay because of the more reliable growth features of NIH 3T3 cells compared with the D56 cell line. For laboratories analyzing clinical materials, this report may prove useful in establishing detection assays for Eco-RCR.

Deregulation of erythropoiesis by the Friend spleen focus-forming virus.

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.

Synergistic effect of human lactoferrin and recombinant murine interferon-gamma on disease progression in mice infected with the polycythemia-inducing strain of the Friend virus complex.

Mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P) have been used as a leukemic mouse model. In the present study, purified iron-saturated human lactoferrin (LF) and recombinant murine (rmu) interferon-gamma (IFN-gamma), alone or in combination, were used to influence disease progression in virally infected mice. DBA/2 mice were injected i.v. with FVC-P, and were treated s.c. with 100 micrograms LF at day 7, and/or rmuIFN-gamma at 5 x 10(4) units/day for 3 days beginning at day 6 after viral infection. Mice were assessed for survival, and also 14 days after virus inoculation, the mice were killed and spleen extracts were assessed for spleen focus forming virus (SFFV) titers by spleen focus forming unit (SFFU) assay, SFFV mRNA and genomic DNA expression, and natural killer (NK) cell activity. Treatment with LF or rmuIFN-gamma alone had little or no effect on SFFU numbers or SFFV mRNA or genomic DNA expression. However, dramatically decreased SFFV titers and levels of SFFV mRNA and genomic DNA were observed in mice treated with the combination of LF and rmuIFN-gamma. NK cell activity decreased by FVC-P was returned to normal levels by LF and rmuIFN-gamma. The combined treatment also enhanced the survival rates of FVC-P-infected mice. The results suggest synergistic suppressive effects of LF with rmuIFN-gamma on disease progression in FVC-P-infected mice. This information might be of significance as a potential therapy for patients with leukemia and those infected with retroviruses.

Activation of the Jun N-terminal kinase pathway by friend spleen focus-forming virus and its role in the growth and survival of friend virus-induced erythroleukemia cells.

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.

Insertion of sequences into the 3' untranslated region of a replication-competent spleen necrosis virus vector disrupts env gene expression.

The genomes of all replication-competent retroviruses contain cis-acting elements that regulate gene expression. However, the identities of many of these elements remain to be characterized. Inserting sequences into the 3' untranslated region of a replication-competent spleen necrosis virus (SNV) vector disrupted its ability to replicate. Inserts varying in sequences and sizes (0.4-kb to 1.6-kb) all resulted in this defect. Genetic compensation experiments and immunostaining revealed that env gene expression was deficient. Northern analysis indicated the presence of spliced viral mRNA of the correct size although at a reduced level compared to a wildtype vector. It is likely that the block in env expression occurs at a post-transcriptional step. These results suggest that the function of a cis-acting element distinct from the constitutive transport element is disrupted by the inserted sequences into the 3' untranslated region of SNV.

Friend virus replication in normal and immunosuppressed C57BL/6 mice.

Young adult C57BL/6 mice are resistant to the replication of Friend virus. We show here that this resistance is not absolute. In 7-week old C57BL/6 mice injected with NB-tropic Friend virus iv, high titers of SFFV could be recovered from the spleen at 8 days after infection but by 21 days, no virus was detectable. A single dose of anti-Thy 1.2 monoclonal antibody iv before FV infection permitted continued replication of SFFV in these animals. This finding strongly supports the hypothesis that SFFV replication in C57BL/6 mice is restricted by a T cell-mediated immune response.

Induction of the autonomous stage of transformation in erythroid cells infected with SFFV: helper virus is not required.

The erythroleukemia induced by the Friend spleen focus-forming virus (SFFV) in mice exemplifies a multistep oncogenic process. Its sequential steps include a rapid polyclonal hyperplastic stage and a more slowly developing malignant stage characterized by autonomous erythroid cells. We report here that the helper virus normally present in mice infected by SFFV is not required for development of the second stage of transformation. In this study, mice were infected with a polycythemia-inducing variant of SFFV which was prepared as a helper-free stock (L. Wolff and S. Ruscetti, 1985, Science 228, 1549). Highly malignant cells could be detected in helper-free SFFV-infected mice by their transplantability into the omentum of sublethally irradiated mice, and erythroleukemia cell lines, typical of previously isolated Friend murine erythroleukemia cell lines, could be established from diseased spleens. Like their helper virus-containing counterparts, the lines established with helper-free SFFV are inducible for hemoglobin synthesis with a variety of chemicals, but not erythropoietin, and express p53, a marker of malignant transformation. Although the cells expressed SFFV encoded proteins, none expressed gene products of replication competent murine leukemia viruses.

Interference established in mice by infection with Friend murine leukemia virus.

Retroviral interference is manifested in chronically infected cells as a decrease in susceptibility to superinfection by virions using the same cellular receptor. The pattern of interference reflects the cellular receptor specificity of the chronically infecting retrovirus and is mediated by the viral envelope glycoprotein, which is postulated to bind competitively all cellular receptors available for viral attachment. We established retroviral interference in mice by infecting them with Friend murine leukemia virus and them measured susceptibility to superinfection by challenging the mice with the erythroproliferative spleen focus-forming virus. Infection of approximately 10% of nucleated splenocytes rendered mice 1% as susceptible to superinfection as untreated controls. The magnitude of this effect was the same in mice incapable of producing neutralizing antibodies or genetically deficient for T cells. The results indicated that retroviral interference in vivo was established rapidly with infection of a fraction of the host cell population and that the decrease in susceptibility to superinfection occurred without a detectable contribution by immunologic factors.

Augmentation of helper virus replication in the presence of defective retrovirus.

The interaction between defective spleen focus-forming virus (SFFV) and helper virus(es) in Friend virus (FV) complex has been assumed to be one-way, with the helper virus complementing SFFV by supplying necessary virion components. To test this assumption the expression of both SFFV and helper virus in partially congenic mice which differ at the Fv-2 locus, a gene that specifically controls susceptibility to SFFV, was analysed. When the mice were infected with LLV (a strain of Friend SFFV-free helper virus), there was no detectable effect of Fv-2 genotype on LLV expression as tested by virus infectivity in the XC plaque assay or by quantitative viral antigen analysis in an immunoprecipitation assay. However, after infection with FV complex there was an amplification of LLV (as well as SFFV) synthesis in Fv-2s as compared with Fv-2r hosts. To determine whether the increased LLV synthesis in Fv-2s mice was due to an increased population of susceptible target cells as a result of SFFV infection and/or transformation, the ratios of LLV-infected cells in the spleens of LLV- and FV-infected Fv-2s hosts in an infectious centre assay, were compared. Since the percentage of LLV-infected cells was equivalent in both instances, the higher rate of LLV synthesis after infection with FV complex was presumably due to intrinsic properties of SFFV-infected erythroid cells.