Recombination between feline leukemia virus subgroup B or C and endogenous env elements alters the in vitro biological activities of the viruses.

Abstract Abstract 4243 Feline Leukemia Virus subgroup C Receptor (FLVCR) was originally identified and cloned as a cell-surface protein receptor for feline leukemia virus subgroup C, causing pure red blood cell aplasia in cats. Recent studies have demonstrated that FLVCR is a heme exporter which is essential for erythropoiesis. The heme efflux via FLVCR was shown to be essential for erythroid differentiation in K562 cells as well as in CD34+ precursors cells1. Moreover, Keel and co-authors have reported that Flvcr-null mice die in utero due to the failure of fetal erythropoiesis; also post-natal mice lacking FLVCR showed severe anemia. In addition to the erythroid defect, Flvcr-null embryos display defective growth and developmental anomalies2. We have identified an alternative transcription start site giving rise to a novel FLVCR isoform (FLVCRb). Flvcr-b transcript completely lacks the first exon of the canonical isoform (FLVCRa) and code for a putative 6 transmembrane domain containing protein ubiquitously expressed. In vitro over-expression of FLVCRa and FLVCRb showed that the two proteins display different subcellular localization. As expected, FLVCRa is localized at the cell membrane while FLVCRb is in the mitochondrial compartment. The mitochondrial localization of this novel isoform is further confirmed by the identification of a N-terminal mitochondrial sorting presequence. The mitochondrion is the site in which heme biosynthesis occurs. Although all the enzymatic reactions involved in heme synthesis are well characterized, how heme is exported to the cytosol is largely unknown. Because of FLVCRa is a heme exporter at the cell membrane, we hypothesized that FLVCRb could be the mitochondrial heme exporter. According to this hypothesis, FLVCRb expression increased following the stimulation of heme biosynthesis in vitro, in correlation with the increase in hemoglobin production. The ability of FLVCRb to bind and export heme out of the mitochondria is still under investigation. To gain insights into the specific roles of the two isoforms, we have generated Flvcr mutant mice different from those previously reported2. Keel and co-author generated a mouse model in which both FLVCRa and FLVCRb have been deleted. In our mouse model, FLVCRa has been specifically deleted while FLVCRb is still expressed (FLVCRa-null mice). Flvcr-a +/− mice were grossly normal, fertile and indistinguishable from their wild-type littermates. When Flvcr-a +/− mice were intercrossed, no Flvcr-a homozygous knock-out newborns were obtained. The analysis of the embryos from timed Flvcr-a +/− intercrosses showed that the Flvcr-a homozygous knock-out genotype was lethal between E14.5 and the birth. E13.5 Flvcr-a-null embryos showed multifocal and extended hemorrhages, visible in the limbs, head and throughout the body wall, as well as subcutaneous edema. Imcomplete vasculogenesis in the Flvcr-a-null embryos was observed at E11.5, a developmental stage in which hemorrhages were not still evident. This suggests that hemorrhages arise from a defect in the development of embryonic vasculature. Moreover, FLVCRa-null embryos showed skeletal abnormalities as demonstrated by Alcian blue-alizarin red staining. Skeletal malformations were evident in the limb where digits did not form properly and in the head where Meckel's cartilage was incomplete. It is interesting to note that this kind of malformations also occurs in Diamond Blackfan Anemia (DBA) patients. Surprisingly, flow cytometric analyses of E14.5 fetal liver cells double-stained for Ter119 (erythroid-specific antigen) and CD71 (transferrin receptor) showed normal erythropoiesis in Flvcr-a-null embryos, in opposition to what occurs in the previously reported Flvcr-null mice2. Taken together, these data demonstrated that FLVCRb is sufficient to support fetal erythropoiesis when the expression of FLVCRa is loss, likely exporting heme out of the mithocondrion for hemoglobin synthesis. Moreover, the loss of FLVCRa leads to incomplete vasculogenesis, hemorrhages and skeletal malformations highlighting new roles of FLVCRa in these processes. 1. Quigley JG et al. Identification of a human heme exporter that is essential for erythropoiesis. Cell 2004 2. Keel SB et al. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008. Disclosures: No relevant conflicts of interest to declare.

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Properties of Feline Leukemia Virus II. In Vitro Labeling of the Polypeptides 1

Journal of Virology ◽

10.1128/jvi.14.3.700-703.1974 ◽

1974 ◽

Vol 14 (3) ◽

pp. 700-703 ◽

Cited By ~ 2

Author(s):

Leland F. Velicer ◽

Donald C. Graves

Keyword(s):

Leukemia Virus ◽

Feline Leukemia Virus

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Retrovirus-induced feline pure red blood cell aplasia: pathogenesis and response to suramin

Blood ◽

10.1182/blood.v77.7.1442.bloodjournal7771442 ◽

1991 ◽

Vol 77 (7) ◽

pp. 1442-1451

Author(s):

JL Abkowitz

Keyword(s):

Blood Cell ◽

Red Blood Cell ◽

Mononuclear Cells ◽

Leukemia Virus ◽

Transcriptase Inhibitor ◽

Feline Leukemia Virus ◽

Erythroid Progenitors ◽

Marrow Cultures

Feline leukemia virus, subgroup C/Sarma (FeLV-C/Sarma) induces pure red blood cell aplasia in cats. Although erythroid (BFU-E and CFU-E) and granulocyte/macrophage (CFU-GM) progenitors are infected with this virus, only erythropoiesis is impaired. Two to 3 weeks before the onset of anemia, CFU-E become undetectable in marrow cultures while earlier erythroid progenitors (BFU-E) persist, suggesting that FeLV-C/Sarma (presumably via its envelope glycoprotein gp70) inhibits the differentiation of BFU-E to CFU-E in vivo. To correlate in vitro observations with the progression of disease, prospective studies were performed in six cats. These studies showed that at the time that the frequencies of CFU-E decreased in marrow cultures, BFU-E no longer responded to hematopoietic growth factor(s), although the responses of CFU-GM were unchanged. In further studies, anemic cats received suramin, a reverse-transcriptase inhibitor with other diverse effects. Within 4 to 14 days, erythropoiesis improved and up to 1,616 CFU-E were detected per 10(5) marrow mononuclear cells. However, progenitor cells remained infected, suggesting that suramin modulated erythroid differentiation without inhibiting progenitor infection. These observations led to the hypothesis that the gp70 of FeLV-C/Sarma impairs BFU-E differentiation by interference with ligand/receptor interactions or signal transduction pathways unique to erythroid cells. Understanding this mechanism should provide insights into the interactions controlling early erythropoiesis.

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Studies in feline long-term marrow culture: hematopoiesis on normal and feline leukemia virus infected stromal cells

Blood ◽

10.1182/blood.v80.3.651.bloodjournal803651 ◽

1992 ◽

Vol 80 (3) ◽

pp. 651-662

Author(s):

ML Linenberger ◽

JL Abkowitz

Keyword(s):

Stromal Cells ◽

Mononuclear Cells ◽

Clonal Selection ◽

Leukemia Virus ◽

Feline Leukemia Virus ◽

Erythroid Progenitors ◽

Gag Protein ◽

Cell Engraftment

To study the effects of feline leukemia virus (FeLV) on the hematopoietic microenvironment, a two-step feline long-term marrow culture (LTMC) system was developed and characterized. The adherent, stromal layer of these cultures is composed of fibroblastoid cells (50% to 80%), macrophages (10% to 30%), fat cells (10% to 20%), and large, polygonal cells that express muscle actin (1% to 2%). When fresh, enriched marrow mononuclear cells (MMNC) were added to 3-week-old irradiated stromal cultures, nonadherent erythroid progenitors (BFU-E) and granulocyte/macrophage progenitors (CFU-GM) could be detected for up to 5 and 12 weeks, respectively. LTMC stromal layers established from marrow cells from cats viremic with either a nonpathogenic strain of FeLV (FeLV-A/61E) or the anemogenic strain FeLV-C/Sarma were morphologically equivalent to uninfected LTMC stromal layers, although more than 80% of the stromal cells expressed FeLV gag protein. When FeLV-infected stromal cultures were recharged with uninfected MMNC, altered patterns of hematopoiesis were observed, compared with recharged, uninfected stromal cultures. In cultures with infected stroma, fewer nonadherent cells (NAC), nonadherent BFU-E, and nonadherent CFU-GM were detected during the first 4 to 5 weeks after recharge. In contrast, greater numbers of NAC and nonadherent CFU-GM were found from weeks 5 to 12 after recharge. When FeLV-infected stromal cultures were recharged with MMNC from a cat heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G-6- PD), the percentage of nonadherent CFU-GM expressing the domestic type G-6-PD isoenzyme remained stable over time (mean % domestic [%d], 53% +/- 3%), and was equivalent to that of nonadherent CFU-GM maintained in uninfected cultures (mean %d, 56% +/- 3%), indicating that clonal drift or clonal selection was not responsible for the enhanced maintenance of CFU-GM. Furthermore, as only 10% to 20% of recharged hematopoietic cells became infected with FeLV in vitro, it is unlikely that the altered pattern was due to progenitor infection. We hypothesize that the increase in NAC and nonadherent CFU-GM in FeLV-infected cultures resulted from enhanced growth factor production by stromal cells. The two-step LTMC system may facilitate the characterization of stromal- derived factors that affect progenitor cell engraftment and proliferation.

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Cell lines produce factors that induce fetal hemoglobin in human BFUe- derived colonies

Blood ◽

10.1182/blood.v80.10.2650.bloodjournal80102650 ◽

1992 ◽

Vol 80 (10) ◽

pp. 2650-2655

Author(s):

P Constantoulakis ◽

M Walmsley ◽

R Patient ◽

T Papayannopoulou ◽

T Enver ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Virus ◽

Fetal Hemoglobin ◽

Feline Leukemia Virus ◽

In Vitro Translation ◽

Expression Cloning ◽

Bladder Cell ◽

Relative Production ◽

Established Cell

Established cell lines were screened for secretion of activities than can stimulate fetal hemoglobin (HbF) production in adult burst-forming unit-erythroid (BFUe) cultures. Conditioned media from four cell lines, a human teratocarcinoma, an osteosarcoma, a bladder cell carcinoma, and feline leukemia virus (FeLV) A-infected feline fibroblasts (FEF-A cells), consistently increased the relative production of fetal globin in BFUe-derived colonies. In vitro translation of RNA from these cells in Xenopus oocytes yielded products that increased the gamma to gamma+beta ratio in adult erythroid colonies. These results demonstrate that a variety of cell lines produce factors that stimulate the production of HbF in vitro. The genes of such factors could be isolated by expression cloning of cDNA from cell lines using the Xenopus oocyte system.

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Host cell range of T-lymphotropic feline leukemia virus in vitro

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2006.05.039 ◽

2006 ◽

Vol 345 (4) ◽

pp. 1466-1470 ◽

Cited By ~ 3

Author(s):

Takayuki Shojima ◽

Risa Nakata ◽

Takayuki Miyazawa

Keyword(s):

Host Cell ◽

Leukemia Virus ◽

Feline Leukemia Virus ◽

Cell Range

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The Role of In Vitro-Induced Lymphocyte Apoptosis in Feline Immunodeficiency Virus Infection: Correlation with Different Markers of Disease Progression

Journal of Virology ◽

10.1128/jvi.72.11.9025-9033.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 9025-9033 ◽

Cited By ~ 18

Author(s):

Edgar Holznagel ◽

Regina Hofmann-Lehmann ◽

Christian M. Leutenegger ◽

Karin Allenspach ◽

Silke Huettner ◽

...

Keyword(s):

Feline Immunodeficiency Virus ◽

Peripheral Blood ◽

Leukemia Virus ◽

Peripheral Blood Lymphocytes ◽

Feline Leukemia Virus ◽

Lymphocyte Apoptosis ◽

Blood Lymphocytes ◽

Immunodeficiency Virus ◽

Induced Apoptosis

ABSTRACT Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4+T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4+ cell count (P = 0.002), bright CD8+ cell count (P = 0.009), and CD4/CD8 ratio (P = 0.01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.

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