Susceptibility of nude mice carrying the Fv-4 gene to Friend murine leukemia virus infection

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). Despite complete resistance to ecotropic MuLV infection in mice carrying the Fv-4 gene, it is known that cells carrying the resistance gene in tissue culture do not always show resistance as extensive as that in vivo (H. Yoshikura and T. Odaka, JNCI 61:461-463, 1978). To investigate the immunological effect on resistance in vivo, we introduced the Fv-4 gene into BALB/c nude mice (Fv-4-/- nude[nu/nu]) by mating them with Fv-4 congenic BALB/c mice (Fv-4r/r nude+/+) and examined the susceptibility of the F2 progeny to F-MuLV. All BALB/c nude mice without the Fv-4 gene (Fv-4-/- nude[nu/nu]) were permissive to F-MuLV and developed erythroleukemia within 2 weeks after virus inoculation. The BALB/c nude mice with the Fv-4 gene (Fv-4r/r nude[nu/nu]) did not develop leukemia, and no or little virus was detected in the spleen 7 weeks after virus inoculation. The resistance to F-MuLV was dominant in (Fv-4 congenic BALB/c x BALB/c nude) F1 mice with the Fv-4r/- nude(nu/+) genotype as strictly as in (Fv-4 congenic BALB/c x BALB/c) F1 mice with the Fv-4r/- nude+/+ genotype. However, almost all BALB/c nude mice with the Fv-4r/- nude(nu/nu) genotype developed the disease within 7 weeks, and the virus was detected in all of their spleens even in the mice without leukemia. These results show that the resistance caused by the Fv-4 gene is recessive in nude mice and dominant in BALB/c mice. Some immunological effects, perhaps cell-mediated immunity, may play important roles in the resistance to F-MuLV infection in vivo in addition to the dosage effect of the Fv-4 product.     

HIV service provision and training needs in outpatient mental health settings

The erythropoietin receptor (EpoR) has been previously shown to contain a cytoplasmic C-terminal negative regulatory domain, experimental deletion or mutation of which leads to increased sensitivity of expressing cells to the effects erythropoietin (Epo). We have studied a naturally occurring C-terminal truncation mutant of the human EpoR by stably transfecting the growth factor-dependent hematopoietic tissue culture cell line 32D with expression plasmids containing either the wildtype or mutant human EpoR cDNA, thus rendering the cells dependent on Epo for viability and proliferation. In Epo dose-response assays, cells expressing the mutant EpoR displayed hyperresponsiveness to Epo compared with cells expressing comparable numbers of the wild-type EpoR cultured in the presence of fetal bovine serum. We investigated whether enhanced Epo sensitivity of cells expressing the truncated EpoR is associated with alteration in Epo receptor-mediated activation of Stat5, which could have a role in Epo-induced proliferation. Although maximal Stat5 activation in response to a given concentration of Epo was comparable in 32D cells expressing the wild-type or truncated EpoRs, the time course of Epo-induced Stat5 activation was very different. Gel-mobility shift studies revealed the presence of Stat5 DNA-binding activity in nuclear and cytoplasmic extracts of cells expressing the truncated EpoR for a significantly longer time than that observed in similar extracts of cells expressing the wild-type EpoR consistent with decreased rate of inactivation of Stat5 in cells expressing the mutant EpoR. Epo-dependent tyrosine phosphorylation of both Stat5 and Jak2 was also substantially prolonged in cells expressing the truncated EpoR. These results suggest a role for Stat5 in regulation of Epo-mediated cell growth and implicate altered kinetics of Epo-induced Jak2 and Stat5 activation in the pathogenesis of familial erythrocytosis associated with this naturally occurring EpoR gene mutation.     

The prolactin receptor rescues EpoR-/- erythroid progenitors and replaces EpoR in a synergistic interaction with c-kit

We recently showed that a retrovirally transduced prolactin receptor (PrlR) efficiently supports the differentiation of wild-type burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors in response to prolactin and in the absence of erythropoietin (Epo). To examine directly whether the Epo receptor (EpoR) expressed by wild-type erythroid progenitors was essential for their terminal differentiation, we infected EpoR-/- progenitors with retroviral constructs encoding either the PrlR or a chimeric receptor containing the extracellular domain of the PrlR and intracellular domain of EpoR. In response to prolactin, both receptors were equally efficient in supporting full differentiation of the EpoR-/- progenitors into erythroid colonies in vitro. Therefore, there is no requirement for an EpoR-unique signal in erythroid differentiation; EpoR signaling has no instructive role in red blood cell differentiation. A synergistic interaction between EpoR and c-kit is essential for the production of normal numbers of red blood cells, as demonstrated by the severe anemia of mice mutant for either c-kit or its ligand, stem cell factor. We show that the addition of stem cell factor potentiates the ability of the PrlR to support differentiation of both EpoR-/- and wild-type CFU-e progenitors. This synergism is quantitatively equivalent to that observed between c-kit and EpoR. Therefore, there is no requirement for an EpoR-unique signal in the synergistic interaction between c-kit and EpoR.     

Characterization of a live-attenuated retroviral vaccine demonstrates protection via immune mechanisms

Live-attenuated retroviruses have been shown to be effective retroviral vaccines, but currently little is known regarding the mechanisms of protection. In the present studies, we used Friend virus as a model to analyze characteristics of a live-attenuated vaccine in protection against virus-induced disease. Highly susceptible mice were immunized with nonpathogenic Friend murine leukemia helper virus (F-MuLV), which replicates poorly in adult mice. Further attenuation of the vaccine virus was achieved by crossing the Fv-1 genetic resistance barrier. The minimum dose of vaccine virus required to protect 100% of the mice against challenge with pathogenic Friend virus complex was determined to be 10(3) focus-forming units of attenuated virus. Live vaccine virus was necessary for induction of immunity, since inactivated F-MuLV did not induce protection. To determine whether immune cells mediated protection, spleen cells from vaccinated donor mice were adoptively transferred into syngeneic recipients. The results indicated that immune mechanisms rather than viral interference mediated protection.     

Effects of ecotropic murine retroviruses on the dual-function cell surface receptor/basic amino acid transporter

The widely expressed Na(+)-independent transporter for basic amino acids (system y+) is the cell surface receptor (ecoR) for ecotropic host-range mouse retroviruses (murine leukemia viruses (MuLVs)), a class of retroviruses that naturally infects only mice or rats. Accordingly, expression of mouse ecoR cDNA in mink CCL64 fibroblasts yields cells (CEN cells) that have y+ transporter activity above the endogenous background and that bind and are infected by ecotropic MuLVs. The effect of ecotropic MuLV infection on expression of y+ transporter was analyzed in mouse and in mink CEN fibroblasts. Chronic infection with ecotropic MuLVs caused 50-70% loss (down-modulation) of mouse y+ transporter in plasma membranes, detected as a reduced Vmax for uptake and outflow of L-[3H]arginine with no effect on Km values. Down-modulation was specific for mouse y+ and did not affect other transporters or the endogenous mink y+, suggesting that it results from specific interaction between mouse y+ and the viral envelope glycoprotein gp70 in the infected cells. Because this partial loss of mouse y+ from cell surfaces is insufficient to explain the complete interference to superinfection that occurs in cells chronically infected with ecotropic MuLVs, alternative explanations for interference are proposed. In contrast to the y+ down-modulation caused by chronic infection, binding of extracellular envelope glycoprotein gp70 at 37 degrees C resulted in noncompetitive inhibition of amino acid import by mouse y+ but had no effect on export through this same transporter or on any transporter properties of mink y+. The effects of gp70 on transport kinetics suggest that it slows the rate-limiting step of the amino acid import cycle, a conformational transition of the empty transporter in which the binding site moves from the inside back to the outside of the cell, and that gp70 has no effect on the rate-limiting step of the amino acid export cycle. Infected cells retain substantial y+ activity. Moreover, the virus binding site on ecoR is in a mobile region that changes conformation during the amino acid transport cycle.     

The distal cytoplasmic domain of the erythropoietin receptor induces granulocytic differentiation in 32D cells

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood 89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)-dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation.     

A small region of the ecotropic murine leukemia virus (MuLV) gag gene profoundly influences the types of polytropic MuLVs generated in mice

The vast majority of recombinant polytropic murine leukemia viruses (MuLVs) generated in mice after infection by ecotropic MuLVs can be classified into two major antigenic groups based on their reactivities to two monoclonal antibodies (MAbs) termed Hy 7 and 516. These groups very likely correspond to viruses formed by recombination of the ecotropic MuLV with two distinct sets of polytropic env genes present in the genomes of inbred mouse strains. We have found that nearly all polytropic MuLVs identified in mice infected with a substrain of Friend MuLV (F-MuLV57) are reactive with Hy 7, whereas mice infected with Moloney MuLV (Mo-MuLV) generate major populations of both Hy 7- and 516-reactive polytropic MuLVs. We examined polytropic MuLVs generated in NFS/N mice after inoculation with Mo-MuLV-F-MuLV57 chimeras to determine which regions of the viral genome influence this difference between the two ecotropic MuLVs. These studies identified a region of the MuLV genome which encodes the nucleocapsid protein and a portion of the viral protease as the only region that influenced the difference in polytropic-MuLV generation by Mo-MuLV and F-MuLV57.     

Recombinant human immunodeficiency Pr55gag virus-like particles presenting chimeric envelope glycoproteins induce cytotoxic T-cells and neutralizing antibodies

Very recently, we demonstrated that the replacement of the human immunodeficiency virus type-1 (HIV-1) gp41 transmembrane protein by an Epstein-Barr virus gp220/350-derived membrane anchor resulted in the incorporation of chimeric envelope (Env) oligomers into Pr55gag virus-like particles (VLPs), exceeding that of wild-type gp160 by a factor of 10. In this study, we examined the immunostimulatory properties of Pr55gag VLPs to both (i) chimeric HIV-1 gp120 external envelope proteins and (ii) full-length gp160 presented on the outer surface of the particles. Immunization studies carried out with VLPs presenting different derivatives of the chimeric and wild-type Env proteins elicited a consistent anti-Pr55gag as well as anti-Env antibody response in complete absence of additional adjuvants. In both cases, the immune sera exhibited an in vitro neutralizing activity against homologous HIV-1 infection in MT4 cells. Noteworthy, these VLPs were also capable of inducing a strong CD8+ cytotoxic T-cell (CTL) response in immunized BALB/c mice that was directed toward a known CTL epitope in the third variable domain V3 of the gp120 external glycoprotein. However, the induction of V3-loop-specific CTLs critically depended on the amounts of Env proteins that were presented by the Pr55gag VLPs. Moreover, the CD8+ CTL response was not significantly altered by adsorbing the VLPs to alum or by repeated booster immunizations. These results illustrate that Pr55gag VLPs provide a safe and effective means of enhancing neutralizing humoral responses to particle-entrapped gp120 proteins and are also capable of delivering these proteins to the MHC class I antigen processing and presentation pathway. Therefore, antigenically expanded Pr55gag VLPs represent an attractive approach in the design of vaccines for which specific stimulation of neutralizing antibodies and cytotoxic effector functions to complex glycoproteins is desired.     

Impaired generation of anti-AKR/Gross murine leukemia virus cytotoxic T lymphocytes in mice experimentally infected with MuLV

C57BL/6 (B6) and C57BL/6.Fv-1n (B6.Fv-1n) mice mount AKR/Gross murine leukemia virus (MuLV)-specific cytolytic T lymphocyte (CTL) responses following primary and secondary stimulation with AKR/Gross MuLV-induced tumor cells. In contrast, mice exposed to infectious virus rather than virus-infected cells generate little, if any, antiviral CTL activity. In this report, we show that inoculation of B6 or B6.Fv-1n mice with MuLV prior to priming with H-2-matched AKR/Gross virus antigen-positive tumor cells resulted in a profound inhibition of the virus-specific CTL response. Antiallogeneic major and minor histocompatibility antigen-specific CTL responses were not significantly diminished in MuLV-infected mice. The AKR/Gross MuLV-specific CTL response in B6 mice was inhibited by NB-tropic (SL3-3NB, Friend and Moloney), but not N-tropic (AKR623) MuLV, suggesting that productive infection of host cells was required. We were unable to inhibit the in vitro generation of virus-specific CTL by adding modulator cells from virus-infected mice to mixed lymphocyte-tumor cell cultures (MLTC) of spleen cells from uninfected animals. We also failed to augment CTL generation in MLTC from virus-infected animals by adding exogenous IL-2 or CD4+ lymphocytes from uninfected, tumor-primed mice. Taken together, the data suggested that the inhibition resulted from either a direct or an indirect effect on the in vivo priming of virus-specific CD8+ cells. It is therefore interesting that MuLV such as Friend and Moloney, which do not encode the immunodominant epitope recognized by anti-AKR/Gross MuLV CTL, are nonetheless able to specifically inhibit this response. These results demonstrate a potentially important mechanism by which retroviruses may escape CTL-mediated immunity.     

Early events in erythropoietin-induced signaling

The cloning of the erythropoietin receptor (EpoR) in 1989 has allowed very rapid progress in our understanding of the early intracellular events that may be triggered by erythropoietin (Epo) in erythroid progenitor cells. From studies carried out primarily with cell lines expressing exogenous wild-type and mutant EpoRs, it appears that the activated EpoR is capable of triggering many of the same cascades that are utilized by receptors possessing endogenous tyrosine kinase domains. The major challenge over the next decade lies in seeing if these same signaling pathways are also utilized by normal Epo-responsive erythroid progenitors, discriminating between proliferation and differentiation inducing events in these cells, and determining whether various hematologic disorders can be attributed to aberrations in these signaling pathways.     

Oxidative stress and interleukins in seminal plasma during leukocytospermia

Various roles for the viral receptor, CD4, have been proposed in facilitating human immunodeficiency virus type 1 (HIV-1) entry, including virion binding to the target cell and the induction of conformational changes in the viral envelope glycoproteins required for the membrane fusion reaction. Here, we compare the structural requirements in the CDR2-like loop of CD4 domain 1, the major contact site of the gp120 envelope glycoprotein, for gp120 binding and virus entry. For every CD4 mutant examined, the level of cell surface expression and the gp120 binding affinity were sufficient to explain the relative ability to function as a viral receptor. The decrease in relative infectibility associated with decreased gp120 binding affinity was more pronounced at lower cell surface CD4 concentrations. These results imply that both receptor density and affinity determine the efficiency of HIV-1 entry and that specific structures in the CD4 residues examined are probably not required for HIV-1 entry functions other than gp120 binding.     

The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.     

Genetic vaccination against the melanocyte lineage-specific antigen gp100 induces cytotoxic T lymphocyte-mediated tumor protection

Melanocyte lineage-specific antigens, such as gp100, have been shown to induce both cellular and humoral immune responses against melanoma. Therefore, these antigens are potential targets for specific antimelanoma immunotherapy. A novel approach to induce both cellular and humoral immunity is genetic vaccination, the injection of antigen-encoding naked plasmid DNA. In a mouse model, we investigated whether genetic vaccination against the human gp100 antigen results in specific antitumor immunity. The results demonstrate that vaccinated mice were protected against a lethal challenge with syngeneic B16 melanoma-expressing human gp100, but not control-transfected B16. Both cytotoxic T cells and IgG specific for human gp100 could be detected in human gp100-vaccinated mice. However, only adoptive transfer of spleen-derived lymphocytes, not of the serum, isolated from protected mice was able to transfer antitumor immunity to nonvaccinated recipients, indicating that CTLs are the predominant effector cells. CTI, lines generated from human gp100-vaccinated mice specifically recognized human gp100. Interestingly, one of the CTL lines cross-reacted between human and mouse gp100, indicating the recognition of a conserved epitope. However, these CTLs did not appear to be involved in the observed tumor protection. Collectively, our results indicate that genetic vaccination can result in a potent antitumor response in vivo and constitutes a potential immunotherapeutic strategy to fight cancer.     

SHP-1 phosphatase C-terminus interacts with novel substrates p32/p30 during erythropoietin and interleukin-3 mitogenic responses

SHP-1 protein tyrosine phosphatase is a critical negative regulator of mitogenic signaling, as demonstrated by the heightened growth responses to hematopoietic growth factors in hematopoietic cells of motheaten mice, which lack functional SHP-1 expression due to mutations in the SHP-1 gene. The mitogenic signaling molecules dephosphorylated by SHP-1 have not been fully identified. We detected two proteins (p32/p30) that are hyperphosphorylated in a DA3/erythropoietin receptor (EpoR) cell line that expresses a mutant containing the SHP-1 C-terminus that suppresses the function of the endogenous phosphatase and induces hyperproliferative responses to interleukin-3 (IL-3) and Epo. Hyperphosphorylated p32/p30 are also detected in motheaten hematopoietic cells, demonstrating an association of p32/p30 hyperphosphorylation with SHP-1-deficiency and growth factor-hyperresponsiveness. The hyperphosphorylated p32/30 associate with SHP-1 via its C-terminus, because they coimmunoprecipitate with the phosphatase and the C-terminal mutant and they bind in vitro to a synthetic peptide of the mutant but not the GST fusion proteins of SHP-1 SH2 domains. Induction of p32/p30 phosphorylation by IL-3 or Epo occurs mainly at 2 to 18 hours poststimulation in the DA3/EpoR cell line, indicating p32/p30 as novel signaling molecules during cell cycle progression. These data demonstrate a function for the SHP-1 C-terminus in recruiting potential substrates p32/p30 and suggest that SHP-1 may regulates mitogenic signaling by dephosphorylating p32/p30.     

Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef

nef genes from two laboratory grown human immunodeficiency virus type 1 (HIV-1) strains and from two proviruses that had not been propagated in vitro were introduced into CD4+ lymphoblastoid CEM cells. The stable expression of all four Nef proteins was associated with an almost complete abrogation of CD4 cell surface localization. The consequences of the presence of Nef on gp160 cleavage, gp120 surface localization, and envelope-induced cytopathic effect were examined in CEM cells in which the HIV-1 env gene was expressed from a vaccinia virus vector. The presence of Nef did not modify the processing of gp160 into its subunits but resulted in a significant decrease of cell surface levels of gp120, associated with a dramatic reduction of the fusion-mediated cell death. Surface levels of mutant envelope glycoproteins unable to bind CD4 were not altered in Nef-expressing cells, suggesting that the phenomenon was CD4 dependent. The intracellular accumulation of fully processed envelope glycoproteins could significantly delay the cytopathic effect associated with envelope surface expression in HIV-infected cells and may be relevant to the selective advantage associated with Nef during the in vivo infectious process.