Budding of retroviruses from polarized epithelial Madin–Darby canine kidney cells (MDCK) takes place specifically at the basolateral membrane surface. This sorting event is suspected to require a specific signal harbored by the viral envelope glycoprotein and it was previously shown that, as for most basolateral proteins, the intracytoplasmic domain plays a crucial role in this targeting phenomenon. It is well known that tyrosine‐based motifs are a central element in basolateral targeting signals. In the present study, site‐directed mutagenesis was used to generate conservative or non‐conservative substitutions of each four intracytoplasmic tyrosines of the human immunodeficiency virus (HIV‐1) envelope glycoprotein. This approach revealed that the membrane‐proximal tyrosine is essential to ensure both the basolateral localization of envelope glycoprotein and the basolateral targeting of HIV‐1 virions. Substitutions of the membrane‐proximal tyrosine did not appear to affect incorporation of envelope glycoprotein into the virions, as assayed by virion infectivity and protein content, nor its capability to ensure its role in viral infection, as determined by viral multiplication kinetics. Altogether, these results indicate that the intracytoplasmic domain of the HIV‐1 envelope glycoprotein harbors a unique, tyrosine‐based, basolateral targeting signal. Such a tyrosine‐based targeting signal may play a fundamental role in HIV transmission and pathogenesis.

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin-or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3 and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3 via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and colocalized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wildtype Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3 , had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

Maturation and release of human immunodeficiency virus type 1 (HIV-1) is targeted at the pseudopod of infected mononuclear cells. However, the intracellular mechanism or targeting signals leading to this polarized viral maturation are yet to be identified. We have recently demonstrated the presence of a functional YXXL motif for specific targeting of HIV-1 virions to the basolateral membrane surface in polarized epithelial Madin-Darby canine kidney cells (MDCK). Site-directed mutagenesis was used to demonstrate that the membrane-proximal tyrosine in the intracytoplasmic tail of the HIV-1 transmembrane glycoprotein (gp41) is an essential component of this signal. In the present study, immunolocalization of viral budding allowed us to establish that this tyrosine-based signal is involved in determining the exact site of viral release at the surface of infected mononuclear cells. Substitution of the critical tyrosine residue was also shown to increase the amount of envelope glycoprotein at the cell surface, supporting previous suggestions that the tyrosine-based motif can promote endocytosis. Although alteration of the dual polarization-endocytosis motif did not affect the infectivity of cell-free virus, it could play a key role in cell-to-cell viral transmission. Accordingly, chronically infected lymphocytes showed a reduced ability to transmit the mutant virus to a cocultivated cell line. Overall, our data indicate that the YXXL targeting motif of HIV is active in various cell types and could play an important role in viral propagation; this may constitute an alternative target for HIV therapeutics and vaccine development.

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1␣, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.

It has been clearly established that the budding of the human immunodeficiency virus (HIV-1), a lentivirus, occurs specifically through the basolateral membrane in polarized epithelial cells. More recently, the signal was assigned to a tyrosine-based motif located in the intracytoplasmic domain of the envelope glycoprotein, as previously observed on various other viral and cellular basolateral proteins. In the present study, expression of human T-cell leukemia virus type 1 (HTLV-1) or Moloney murine leukemia virus envelope glycoproteins was used for trans-complementation of an envelope-negative HIV-1. This demonstrated the potential of oncornaviral retrovirus envelope glycoproteins to confer polarized basolateral budding in epithelial Madin-Darby canine kidney cells (MDCK cells). Site-directed mutagenesis confirmed the importance of a common motif encompassing at least one crucial membrane-proximal intracytoplasmic tyrosine residue. The conservation of a similar basolateral maturation signal in different retroviruses further supports its importance in the biology of this group of viruses.

Hemopoietic lineage switch (Hls) 5 and 7 were originally isolated as genes upregulated during an erythroid-to-myeloid lineage switch. We have shown previously that Hls7/Mlf1 imposes a monoblastoid phenotype on erythroleukemic cells. Here we show that Hls5 impedes erythroid maturation by restricting proliferation and inhibiting hemoglobin synthesis; however, Hls5 does not influence the morphology of erythroid cells. Under the influence of GATA-1, Hls5 relocates from cytoplasmic granules to the nucleus where it associates with both FOG-1 and GATA-1. In the nucleus, Hls5 is able to suppress GATA-1-mediated transactivation and reduce GATA-1 binding to DNA. We conclude that Hls5 and Hls7/Mlf1 act cooperatively to induce biochemical and phenotypic changes associated with erythroid/myeloid lineage switching. IntroductionThe erythroblastoid J2E cell line responds to erythropoietin (Epo) by morphologic maturation and hemoglobin synthesis. 1 However, on rare occasions, these cells have undergone a spontaneous lineage switch, and display features of monoblastoid cells, which do not respond to Epo. 2 Thus, regulated expression of structural/ functional genes involved in commitment to the erythroid lineage can be breached, as previously demonstrated with B cell to macrophage lineage switching. 3 Hls5 and Hls7 were isolated as genes markedly up-regulated in the J2E monoblastoid variants. 2,4 Hls7 is the murine orthologue of Myeloid Leukemia Factor 1 (Mlf1), a gene involved in the t(3;5), associated with acute myeloid leukemia. 5 Importantly, ectopic expression of Hls7/Mlf1 in J2E cells imposes a dramatic phenotypic change on the cells, rendering them monoblastoid. 2 Hls5 is a recently identified member of the RING finger, B box, coiled coil (RBCC) 4 or Tripartite motif (TRIM) family, 6 which includes PML, a gene involved in acute promyelocytic leukemia. Hls5 is expressed in a wide variety of hemopoietic cell types, including fetal liver progenitors. 4 The role of Hls5 in erythroid maturation was investigated in this study. MethodsCell lines were cultured with or without Epo, as described previously. 1,2 Assays for differentiation, cell-cycle progression, clonogenicity, confocal microscopy, cytocentrifugation, and staining have been detailed elsewhere, 1,2,4 as have methylcellulose colony assays. 1,2,7 Immunoblotting, immunoprecipitation, and in vitro pulldown assays were used as reported previously. 1,2 Yeast 2 hybrid analyses were described by Ingley et al, 7 and electrophoretic mobility shift assays by Spadaccini et al. 8 The M1␣ construct was used to determine GATA-1 activity, 9 while chromatin immunoprecipitation experiments used primers for the ␤ maj globin promoter and HS2 regions of the ␤-globin locus. 10 Results and discussionTo determine the effects of elevated Hls5 on normal erythroid progenitors, fetal liver cells were infected with retroviruses expressing wild-type Hls5, myc-tagged Hls5 or empty vector control. Methylcellulose assays revealed that both burst forming unitserythroid (BFU-E) and colony forming units...

Through the sequencing of a 42kb cosmid clone we describe a new gene, designated PEREC1, located approximately 1.5kb centromeric of the human apolipoprotein (APO) E-C2 cluster. The combination of dotplot analysis, predicted coding potential and interrogation of the Expressed Sequence Tag (EST) database determined the genomic organisation of PEREC1. Sequence alignment with multiple overlapping ESTs confirmed the predicted splice sites. The predicted cDNA and amino acid sequences of PEREC1 have extensive similarity to the Caenorhabditis elegans protein, C18E9.6. Conserved structural and functional motifs have been defined by combining nucleotide and amino acid analyses to identify third base degeneracy and therefore selection at the protein level. The Poliovirus Receptor Related Protein2 gene (PRR2), previously mapped to chromosome 19q13.2 by Fluorescent In-Situ Hybridisation, has also been located approximately 17kb centromeric of APO E.