Lentivirus degradation and DC-SIGN expression by human platelets and megakaryocytes.

BACKGROUND AND AIM: As platelets are able to endocytose human immunodeficiency virus (HIV), we have investigated the fate of lentiviruses when endocytosed by human platelets and megakaryocytes (MK), and have characterized a specific receptor directly involved in this function. METHODS: Genetically modified (non-replicative) lentiviruses with an HIV envelope (HIV-e) or with a vesicular stomatitis virus protein G envelope (VSV-e) were alternatively used and their interaction with platelets and MK analyzed by electron microscopy (EM) and immunoEM. RESULTS: When incubated with platelets, HIV-e and VSV-e lentiviruses were internalized in specific endocytic vesicles and trafficked to the surface connected canalicular system (SCCS). Double immunolabeling for the viral P24 core protein and alpha-granule markers showed that lentiviruses were degraded in the SCCS after contact with alpha-granule proteins. In culture MK, lentiviruses were found in endocytic vesicles and accumulated in acid phosphatase-containing multivesicular bodies (MVB). The expression of the pathogen receptor dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN) was then demonstrated in platelets by flow cytometry, immunoEM and Western blot. Anti-DC-SIGN antibodies decreased HIV-e lentivirus internalization by platelets, showing that the receptor is functional. Specific signals for DC-SIGN protein and mRNA were also found in MK. CONCLUSION: This study indicates that platelets and MK can internalize lentiviruses in a pathway, which either provide a shelter to lentiviral particles or alternatively disrupts viral integrity. The receptor DC-SIGN is involved in this function.

Molecular study of the hematopoietic zinc finger gene in three unrelated families with gray platelet syndrome.

Hematopoietic zinc finger (HZF) null mice have features reminiscent of patients with gray platelet syndrome (GPS), a rare inherited bleeding disorder. This similarity has suggested that HZF deregulation might be involved in the human disease. The sequence of the eight exons of the HZF gene as well as the study of its expression in blood samples from five patients belonging to three different families did not reveal any modifications when compared with healthy donors. This study indicates that HZF is unlikely to be responsible for GPS.

Identification, phylogeny, and evolution of retroviral elements based on their envelope genes.

Phylogenetic analyses of retroviral elements, including endogenous retroviruses, have relied essentially on the retroviral pol gene expressing the highly conserved reverse transcriptase. This enzyme is essential for the life cycle of all retroid elements, but other genes are also endowed with conserved essential functions. Among them, the transmembrane (TM) subunit of the envelope gene is involved in virus entry through membrane fusion. It has also been reported to contain a domain, named the immunosuppressive domain, that has immunosuppressive properties most probably essential for virus spread within the host. This domain is conserved among a large series of retroviral elements, and we have therefore attempted to generate phylogenetic links between retroviral elements identified from databases following tentative alignments of the immunosuppressive domain and adjacent sequences. This allowed us to unravel a conserved organization among TM domains, also found in the Ebola and Marburg filoviruses, and to identify a large number of human endogenous retroviruses (HERVs) from sequence databases. The latter elements are part of previously identified families of HERVs, and some of them define new families. A general phylogenetic analysis based on the TM proteins of retroelements, and including those with no clearly identified immunosuppressive domain, could then be derived and compared with pol-based phylogenetic trees, providing a comprehensive survey of retroelements and definitive evidence for recombination events in the generation of both the endogenous and the present-day infectious retroviruses.

ERV-L elements: a family of endogenous retrovirus-like elements active throughout the evolution of mammals.

We have previously identified in the human genome a family of 200 endogenous retrovirus-like elements, the HERV-L elements, disclosing similarities with the foamy retroviruses and which might be the evolutionary intermediate between classical intracellular retrotransposons and infectious retroviruses. Southern blot analysis of a large series of mammalian genomic DNAs shows that HERV-L-related elements-so-called ERV-L-are present among all placental mammals, suggesting that ERV-L elements were already present at least 70 million years ago. Most species exhibit a low copy number of ERV-L elements (from 10 to 30), while simians (not prosimians) and mice (not rats) have been subjected to bursts resulting in increases in the number of copies up to 200. The burst of copy number in primates can be dated to shortly after the prosimian and simian branchpoint, 45 to 65 million years ago, whereas murine species have been subjected to two much more recent bursts (less than 10 million years ago), occurring after the Mus/Rattus split. We have amplified and sequenced 360-bp ERV-L internal fragments of the highly conserved pol gene from a series of 22 mammalian species. These sequences exhibit high percentages of identity (57 to 99%) with the murine fully coding MuERV-L element. Phylogenetic analyses allowed the establishment of a plausible evolutionary scheme for ERV-L elements, which accounts for the high level of sequence conservation and the widespread dispersion among mammals.

Cloning of a new murine endogenous retrovirus, MuERV-L, with strong similarity to the human HERV-L element and with a gag coding sequence closely related to the Fv1 restriction gene.

We had previously identified a new family of human endogenous retrovirus-like elements, the HERV-L elements (human endogenous retrovirus with leucine tRNA primer), whose pol gene was most closely related to that of the foamy viruses. HERV-L pol-related sequences were also detected in other mammalian species. The recent cloning of the mouse Fv1 gene (S. Best, P. Le Tissier, G. Towers, and J. P. Stoye, Nature 382:826-829, 1996) has shed light on another HERV-L domain--identified as a gag gene based on its location within the provirus--which was found to be 60% identical, at the nucleotide level, to the Fv1 open reading frame (ORF). We have now cloned the murine homolog of HERV-L which, in contrast to HERV-L, displays fully open reading frames in the gag and pol genes. Its predicted Gag protein shares 43% identity with the Fv1 ORF product. Moreover, the characteristic major homology region of the capsid subdomain can be identified within both proteins, thus strongly emphasizing the gag-like origin of Fv1.

Functional regions of the mouse thrombopoietin receptor cytoplasmic domain: evidence for a critical region which is involved in differentiation and can be complemented by erythropoietin.

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. To identify functionally important regions in the cytoplasmic domain of the TPO receptor, mpl, we introduced wild-type mpl and deletion mutants of murine mpl into the granulocyte-macrophage colony-stimulating factor (GM-CSF)- or erythropoietin (EPO)-dependent human cell line UT7. TPO induced differentiation of UT7-Wtmpl cells, not parental UT7 cells, along the megakaryocytic lineage, as evidenced by decreased proliferation, changes in cell morphology, and increased surface expression and mRNA levels of megakaryocytic markers CD41, CD61, and CD42b. When UT7-mpl cells were cultured long-term in EPO instead of GM-CSF, the TPO effect was dominant over that of EPO. Moreover, the differentiation induced by TPO was more pronounced for cells shifted from EPO to TPO than for cells shifted from GM-CSF to TPO, as shown by the appearance of polyploid cells. Mutational analysis of the cytoplasmic domain of mpl showed that proliferation and maturation functions of mpl can be uncoupled. Two functional regions were identified: (i) the first 69 amino acids comprising the cytokine receptor motifs, box I and box 2, which are necessary for both TPO-induced mitogenesis and maturation; and (ii) amino acids 71 to 94, which are dispensable for proliferation but required for differentiation. Surprisingly, however, EPO could complement this latter domain for TPO-induced differentiation, suggesting a close relationship between EPO and TPO signaling.

Signal transduction by the receptors for thrombopoietin (c-mpL) and interleukin-3 in hematopoietic and nonhematopoietic cells.

Antisense oligonucleotide to the translation initiation sequence of human c-mpI reduced the proliferation of human CD34+ bone marrow cells in response to interleukin-3 (IL-3) alone or to the combination of IL-3 and thrombopoietin (TPO). To investigate the molecular basis for these cytokine interactions, we analyzed the relationship between the receptor subunits for IL-3 and TPO and determined whether both receptors activate identical signal transduction pathways. The function of the receptor subunits was characterized in transiently transfected hepatoma cells and fibroblasts by the activation of gene expression via specific regulatory elements and by the stimulation of DNA-binding activity of STAT proteins. Although c-mpl and IL-3 receptor (IL-3R) reconstituted a qualitatively comparable gene regulatory response, there was no detectable functional interaction between their respective receptor subunits. By comparing the receptor action in different cell lines, we observed that in human hepatoma cells the signaling of c-mpI was 100-fold less sensitive to TPO than in rat hepatoma cells. However, IL-3R signaling was comparable between the two cell types, suggesting that c-mpI and IL-3R do not use identical signal transducing mechanisms. The cytoplasmic domains necessary for c-mpI signaling were determined by testing deletion mutants. The membrane-proximal box 1 sequence motif was critical for gene regulation and for STAT protein activation that seemed to involve the Janus kinase 2 (JAK2). Because IL-3R was less dependent on JAK2 than c-mpI, different levels of JAK2 expression may account, in part, for the quantitative difference in IL-3 and TPO response among various cell lines.

Thrombopoietin activates a STAT5-like factor in hematopoietic cells.

Thrombopoietin (TPO) is a newly cloned cytokine which is the major regulator of circulating platelet levels, acting on both proliferation and differentiation of megakaryocytes. We have investigated the ability of TPO to activate the JAK/STAT pathway in megakaryocytic cell lines. We used either the granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or erythropoietin (EPO)-dependent UT7 cell line in which the murine TPO receptor (mumpl) had been transfected (mumpl-UT7 transfectants) or the MO7E and DAMI cells which express endogenous human TPO receptors. We demonstrated that TPO activates the kinase JAK2 and a STAT5-like transcriptional factor but not STAT1, STAT2, STAT3 or STAT4, in a very rapid and transient manner. In order to better ascertain the specificity of the activation of STAT5-related factor by TPO, we investigated the effect of other cytokines/growth factors. Both GM-CSF and EPO activated the STAT5-like factor. In contrast, neither interferon (IFN)-gamma nor the mitogenic stem cell factor (SCF) activated STAT5, although IFN-gamma did activate STAT1 in those cells. The hematopoietic DNA binding activity related to STAT5 was identified as a p97 tyrosine-phosphorylated protein band which exhibited identical gel mobility to the mammary STAT5. Because v-mpl, a truncated form of the TPO receptor c-mpl, was shown to be oncogenic, we tested the activity of v-mpl on STAT5 and found STAT5 constitutively activated in two different v-mpl-expressing cells, the transiently transfected Cos7 cells and the stable v-mpl-UT7 transfectants. Overall, our data indicate that STAT5 is widely expressed in hematopoietic cells and activated by a number of cytokines, including TPO, GM-CSF and EPO, but not by IFN-gamma or SCF.

Constitutive activation of a variant of the env-mpl oncogene product by disulfide-linked homodimerization.

The myeloproliferative leukemia retrovirus (MPLV) has the v-mpl cellular sequences transduced in frame with the deleted and rearranged Friend murine leukemia virus env gene. The resulting env-mpl fusion oncogene is responsible for an acute myeloproliferative disorder induced in mice by MPLV. v-mpl is a truncated form of the c-mpl gene which encodes the receptor for thrombopoietin. We investigated the contribution of the Env-Mpl extracellular domain in the constitutive activation of this truncated cytokine receptor and found that the rearrangement of the env sequences in the env-mpl fusion gene was not required for oncogenicity. A pathogenic variant, DEL3MPLV, was generated, which differs from MPLV by the deletions of 22 amino acids of the Env signal peptide, all of the mature Env sequences, and 18 N-terminal amino acids of the v-Mpl extracellular domain. The resulting del3-mpl oncogene product conserves in its extracellular region the first 12 amino acids of the Env signal sequence including a cysteine residue, and 25 amino acids of the v-Mpl. We show here that a mutation converting this cysteine to a glycine completely abolishes del3-mpl oncogenicity and that the del3-mpl oncogene product is constitutively activated by disulfide-linked homodimerization.

Characterization of mpl cytoplasmic domain sequences required for myeloproliferative leukemia virus pathogenicity.

v-mpl is a truncated form of a receptor-like chain which belongs to the cytokine receptor superfamily. This sequence has been transduced in the myeloproliferative leukemia virus as an env-mpl fusion gene responsible for an acute myeloproliferative disorder in mice. We constructed a series of viral mutants in the mpl sequence. Analysis of their oncogenic potential in vivo indicated that a critical 69-amino-acid-long cytoplasmic domain of v-Mpl is required for myoproliferative leukemia virus pathogenicity. We also developed an in vitro assay and showed that expression of the env-mpl gene confers growth factor independence to murine as well as to human hematopoietic growth factor-dependent cell lines. These findings strongly suggest that v-Mpl delivers a constitutive proliferative signal through a limited region of its cytoplasmic domain.

The 'WS motif' common to v-mpl and members of the cytokine receptor superfamily is dispensable for myeloproliferative leukemia virus pathogenicity.

Several motifs are conserved in the extracellular domain of the cloned chains of the recently described cytokine receptor superfamily. One of them, usually close to the transmembrane region, is the 'WS motif'. Its function remains unknown, but it has been recently shown that the integrity of this motif is essential for interleukin 2 receptor beta-chain and erythropoietin receptor activity [Miyazaki, T., Maruyama, M., Yamada, G., Hatakeyama, M. & Taniguchi, T. (1991). EMBO J., 10, 3191-3197; Watowich, S.S., Yoshimura, A., Longmore, G.D., Hilton, D.J., Hoshimura, Y. & Lodish, H.R. (1992). Proc. Natl. Acad. Sci. USA, 89, 2140-2144]. This WS motif is present in the v-mpl oncogene, which has been transduced in the myeloproliferative leukemia virus (MPLV). v-mpl encodes a truncated transmembrane protein that belongs to this growth factor receptor family. We demonstrate that determinants of MPLV pathogenesis are encoded by the env-mpl fusion gene and that the complete deletion of the WS motif does not abolish MPLV oncogenic properties.