Prevalence of xenotropic murine leukemia virus-related virus infection in different risk populations in Spain.

Human infection with the xenotropic murine leukemia virus-related virus (XMRV) has been associated controversially with prostate cancer and chronic fatigue syndrome. Information is lacking about the mechanisms of transmission and potential risk groups for XMRV infection. Plasma and peripheral blood mononuclear cells (PBMCs) from individuals with retroviral infections, chronic viral hepatitis, autoimmune diseases, prostate cancer, chronic fatigue syndrome, and blood donors were tested for XMRV markers. Antibodies to XMRV proteins p15E and gp70 were examined using research assays. DNA extracted from PBMCs was tested for the presence of XMRV gag and env sequences. A total of 1103 specimens belonging to individuals with chronic fatigue syndrome and/or fibromyalgia (437), prostate cancer (69), HIV-1 (149), HTLV-1/2 (31), chronic hepatitis B (81), chronic hepatitis C (72), autoimmune diseases (18), and blood donors (246) were examined. Overall, three samples (0.3%) were p15E seroreactive (two HTLV-1 and one HCV patient). Another 15 (1.4%) were gp70 seroreactive (six chronic fatigue syndrome-fibromyalgia, four blood donors, two HIV-1, one prostate cancer, one HBV, and one HCV). Four specimens were initially positive for XMRV gag sequences, but none could be confirmed by repeated testing. In summary, no evidence of XMRV infection was found in populations with retroviral and viral hepatitis infections in Spain. Likewise, XMRV was not recognized in patients with autoimmune diseases, chronic fatigue syndrome-fibromyalgia, prostate cancer, or healthy blood donors.

Clonagem e expressão da glicoproteína transmembrana do vírus linfotrópico de células T humanas em sistema procarioto / Cloning and expression of the transmembranic glycoprotein from human T cell lymphotropic virus in a prokaryotic system

O HTLV-1 é o vírus causador da leucemia/linfoma de célula T no adulto e de uma desordem neurológica conhecida por mielopatia associada ao HTLV ou paraparesia espástica tropical. Um dos modos de transmissão é pelo sangue contaminado e seus subprodutos e, devido ao risco de infecções associadas ao HTLV sua pesquisa na triagem de doadores de sangue foi introduzida no Brasil a partir de 1993. Os kits diagnósticos utilizados nos bancos de sangue nacionais são na sua maioria comprados de empresas estrangeiras. O Brasil não detém a tecnologia para produção deste material e há a necessidade de produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, mostramos a expressão da gp21/HTLV-1 em Escherichia coli e sua reatividade frente a anticorpos monoclonais e de pacientes infectados. Expressar tais proteínas é o primeiro passo para obtenção de conjuntos diagnósticos com tecnologia brasileira.

HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology.

Modulation of human neutrophil responses to CD32 cross-linking by serine/threonine phosphatase inhibitors: cross-talk between serine/threonine and tyrosine phosphorylation.

The interplay between serine/threonine and tyrosine phosphorylation was studied in human neutrophils. The direct effects of calyculin and okadaic acid, potent inhibitors of PP1 and PP2A serine/threonine phosphatases, on the patterns of neutrophil phosphorylation, and their effects on the responses of neutrophils to CD32 cross-linking were monitored. After a 2-min incubation with 10-6 M calyculin, a transient tyrosine phosphorylation of a subset of proteins, among which Cbl and Syk, was observed. After a longer incubation (>5 min) with calyculin, concomitant with an accumulation of serine and threonine phosphorylation, neutrophil responses to CD32 cross-linking were selectively altered. Tyrosine phosphorylation of Cbl in response to CD32 cross-linking was inhibited by calyculin, and this inhibition was linked with a slower electrophoretic mobility of Cbl as a consequence of its phosphorylation on serine/threonine residues. However, tyrosine phosphorylation of Syk and of the receptor itself were not affected. Furthermore, the mobilization of intracellular calcium stimulated by CD32 cross-linking was totally abrogated by calyculin. Finally, the stimulation of superoxide production observed in response to CD32 cross-linking was enhanced in calyculin-treated cells. These results suggest that serine/threonine phosphorylation events regulate the signaling pathways activated by CD32 cross-linking in neutrophils and identify a novel mechanism of modulation of the functional responsiveness of human neutrophils to CD32 cross-linking.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins.

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-A resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Two types of HTLV-1 particles are released from MT-2 cells.

The MT-2 cell line transformed by human T-cell leukemia virus type 1 (HTLV-1) contains one complete provirus and seven defective proviruses. Four defective genomes have an identical structure (LTR-MA-deltaCA-pX-LTR) with an open reading frame that spans from MA to pX, giving rise to a 3.4-kb (24S) RNA transcript encoding a chimeric Gag-pX protein, p28. MT-2 cells release two distinct types of virions. The major "classic" type of particle has a buoyant density of 1.155-1.16 g/cm3 and contains the standard HTLV-I structural proteins and reverse transcriptase (RT). In addition, about 5% of particles are "light," approximately 1.12 g/cm3, and contain p28, RT activity, and the 3.4-kb RNA transcript. RT-PCR and in vitro translation indicate that some of the classic HTLV-1 particles package 3.4-kb RNA as well as full-length 8.5-kb RNA. In addition to matrix features, the p28 protein has a motif resembling a zinc finger at the C-terminal, pX0 region, which may play a role in the assembly of the defective light virions.

Expression of bovine leukemia virus ENV glycoprotein in insect cells by recombinant baculovirus.

The gp51-p30 glycoprotein constituting BLV envelope was expressed in Sf-21 insect cells by means of recombinant baculoviruses. Post-infection cell lysates were analyzed, in order to define the immunologic reactivity of recombinant products. Oligosaccharide chains, containing N-acetylglucosamine, mannose, galactose and sialic acid were found on recombinant gp51-p30. In order to investigate the timing of transcription and translation of the glycoprotein, kinetic assays were carried out on cell lysates and directly in situ on Sf-21 cells during the course of baculovirus infection. The use of different solubilizing reagents was also evaluated in order to rescue recombinant glycoprotein from its subcellular location.

Presence of antibodies to p21X and/or p27rex proteins in sera from human T-cell leukemia virus type I-infected individuals.

The human T-cell leukemia virus type I (HTLV-I) pX gene encodes three nonstructural proteins, p40tax, p27rex and p21X. So far, natural antibodies to p27rex and/or p21X have not been found in sera from HTLV-I-infected individuals, although antibodies to p40tax have been found. Recently, the viral transcripts specific for these proteins were detected in fresh peripheral blood mononuclear cells from HTLV-I-infected individuals by the polymerase chain reaction coupled to reverse transcription, showing the in vivo expression of these proteins. We detected antibodies to p21X and p27rex by an enzyme-linked immunosorbent assay (ELISA) system using a recombinantly produced p21X protein as a common antigen, because p21X is identical to the C-terminal portion of p27rex. The sensitivity of the ELISA was determined to be approximately 100 times greater than that of Western blotting. From the analyzed sera of 31 ATL patients, 30 asymptomatic carriers, 18 HAM patients and 100 healthy donors, three specimens from one ATL patient and two carriers were found to be positive for anti-p21X/p27rex antibodies. The specificity of the ELISA reaction was confirmed by the competitive ELISA test with the highly purified recombinant p21X protein. As of result, we first determined the presence of anti-p21X/p27rex antibodies in a small percentage (3.8%) of the sera from HTLV-I-infected individuals. Even sera from the ATL patients, whose fresh PBMCs contained the transcripts for these proteins, were not found to contain these antibodies, suggesting that the immune response to these proteins is low in HTLV-I-infected humans.

Nerve growth factor stimulates the tyrosine phosphorylation of endogenous Crk-II and augments its association with p130Cas in PC-12 cells.

The cellular homologs of the v-Crk oncogene product consist primarily of Src homology region 2 (SH2) and 3 (SH3) domains. v-Crk overexpression causes cell transformation and elevation of tyrosine phosphorylation in fibroblasts and accelerates differentiation of PC-12 cells in response to nerve growth factor (NGF). To further explore the role of Crk in NGF-induced PC-12 cell differentiation, we found that both NGF and epidermal growth factor stimulate the tyrosine phosphorylation of endogenous Crk II. Moreover, hormone stimulation enhanced the specific association of Crk proteins with the tyrosine-phosphorylated p130Cas, the major phosphotyrosine-containing protein in cells transformed with v-Crk. This interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. Furthermore, the Crk-SH2 domain binds tyrosine-phosphorylated paxillin, a cytoskeletal protein, following treatment of PC-12 cells with NGF or epidermal growth factor. These data suggest that Crk functions in a number of signaling processes in PC-12 cells.

Crkl is complexed with tyrosine-phosphorylated Cbl in Ph-positive leukemia.

The deregulated tyrosine kinase activity of the Bcr/Abl protein has been causally linked to the development of Philadelphia (Ph) chromosome-positive leukemia in mice and man. Abnormally tyrosine-phosphorylated substrates of the Bcr/Abl kinase in Ph-positive cells are likely to contribute to leukemogenesis by interfering with normal signal transduction pathways. We have previously shown that the adaptor molecule Crkl is a major in vivo substrate for the Bcr/Abl tyrosine kinase, and it is thought to connect Bcr/Abl with downstream effectors. In the current study, a tyrosine-phosphorylated protein with a molecular mass of approximately 120 kDa was identified which binds only to the Crkl Src homology 2 (SH2) domain in cells, including Ph-positive patient material, containing an active Bcr/Abl protein. We demonstrate here that this protein is Cbl, originally discovered as an oncogene which induces B-cell and myeloid leukemias in mice. The Crkl SH2 domain binds specifically to Cbl. The Src homology 3 (SH3) domains of Crkl do not bind to Cbl, but do bind Bcr/Abl. These findings suggest the existence of a trimolecular complex involving Bcr/Abl, Crkl, and Cbl and are consistent with a model in which Crkl mediates the oncogenic signal of Bcr/Abl to Cbl.

One-step magnetic purification of recombinant DNA-binding proteins using magnetizable phosphocellulose.

Magnetizable solid phase technology was used to develop a method for the rapid purification of recombinant proteins expressed in Escherichia coli. We describe the purification of two recombinant DNA-binding proteins: the minimal DNA-binding domain of the oncoprotein Myb and full-length yeast TFIIIA. Both were purified in one step directly from an E. coli lysate by means of magnetizable phosphocellulose particles (PhosphoMagnaCel). All operations were performed in microcentrifuge tubes and could be completed within 15 min. High purity and excellent recovery of proteins active in sequence specific DNA-binding were obtained. The procedure allowed the simultaneous purification of eight mutant Myb-proteins within 30 min.

Characterization, partial purification, and peptide sequencing of p130,the main phosphoprotein associated with v-Crk oncoprotein.

The transforming gene v-crk found in CT10 and ASV-1 avian sarcoma viruses induces marked phosphorylation of several proteins in cells expressing p47v-crk (v-Crk). In this work, the main tyrosine-phosphorylated proteins in ASV-1-infected chicken cells and v-crk-transfected rat cells were characterized biochemically. Both these proteins have a molecular mass of about 130 kDa and are tightly associated with v-Crk in vivo. Two-dimensional gel electrophoresis revealed that they are both essentially single proteins (p130) with modifications that result in a broad spot in an acidic region. The broad band of semi-purified p130 became sharp at an elevated position in the gel upon treatment with orthovanadate in vivo or with c-Src kinase produced using a baculovirus vector in vitro, whereas it shifted at a lower position upon treatment with alkaline phosphatase in vitro. These results suggest multiple phosphorylation states of p130, which result in a broad band of p130. Two procedures of immunoaffinity purification were used to purify p130 from 3Y1 cells transfected with v-crk. Approximately 30 pmol of purified p130 was obtained in an immobilized form on a filter starting from 3 x 10(10) cells. Peptide mapping of p130 digested in situ by peptidase revealed that the purity and quantity of the final material were enough for peptide sequencing. Several stretches of partial amino acid sequences were determined, and they indicated that p130 is a novel protein.

The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex.

The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extracellular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction. Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOH-terminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN.

Identification of a C/EBP-Rel complex in avian lymphoid cells.

Protein-protein interactions between the CCAAT box enhancer-binding proteins (C/EBP) and the Rel family of transcription factors have been implicated in the regulation of cytokine gene expression. We have used sequence-specific DNA affinity chromatography to purify a complex from avian T cells that binds to a consensus C/EBP motif. Our results provide evidence that Rel-related proteins are components of the C/EBP-DNA complex as a result of protein-protein interactions with the C/EBP proteins. A polyclonal antiserum raised against the Rel homology domain of v-Rel and antisera raised against two human RelA-derived peptides specifically induced a supershift of the C/EBP-DNA complex in mobility shift assays using the affinity-purified C/EBP. In addition, several kappa B-binding proteins copurified with the avian C/EBP complex through two rounds of sequence-specific DNA affinity chromatography. The kappa B-binding proteins are distinct from the C/EBP proteins that directly contact DNA containing the C/EBP binding site. The identification of a protein complex that binds specifically to a consensus C/EBP site and contains both C/EBP and Rel family members suggests a novel mechanism for regulation of gene expression by Rel family proteins.

Tyrosine kinase activity may be necessary but is not sufficient for c-erbB1-mediated tissue-specific tumorigenicity.

Expression of mutant avian c-erbB1 genes results in tissue-specific transformation in chickens. Site-directed mutagenesis was used to generate kinase-defective mutants of several tissue-specific v-erbB transforming mutants by replacement of the ATP-binding lysine residue in the kinase domain with an arginine residue. These kinase-defective v-erbB mutants were analyzed for their in vitro and in vivo transforming potentials. Specifically, kinase-defective mutants of erythroleukemogenic, hemangioma-inducing, and sarcomagenic v-erbB genes were assessed for their oncogenic potential. In vitro transformation potential was assessed by soft-agar colony formation in primary cultures of chick embryo fibroblasts (CEF). In vivo transformation potential was determined by infection of 1-day-old line 0 chicks with concentrated recombinant retrovirus and then monitoring of birds for tumor formation. These transformation assays demonstrate that kinase activity is absolutely essential for transformation by tissue-specific transforming mutants of the avian c-erbB1 gene. Since all of the tissue-specific v-erbB mutants characterized to date exhibit tyrosine kinase activity in vitro but do not transform all tissues in which they are expressed, we conclude that v-erbB-associated tyrosine kinase activity may be necessary but is not sufficient to induce tumor formation.

Inhibition of v-raf-dependent c-fos expression and transformation by a kinase-defective mutant of the mitogen-activated protein kinase Erk2.

Receptor-bound growth factors elicit intracellular signals that lead to the phosphorylation and activation of numerous intracellular kinases and transcription factors with consequent changes in patterns of gene expression. Several oncogene products are able to mimic these signals, resulting in cell transformation and proliferation. For example, the introduction of oncogenic forms of Raf-1 kinase into fibroblasts induces transformation and leads to the constitutive expression of, among others, the c-fos proto-oncogene. Here it is shown that the elevation of c-fos promoter activity brought about by v-raf is mediated by TCF/Elk-1, which forms a ternary complex with SRF at the serum response element and is a substrate for mitogen-activating protein kinases in vitro. In NIH 3T3 fibroblasts, v-raf activates Erk2, and overexpression of an interfering mutant of Erk2 both blocks the ability of v-raf to activate the c-fos promoter and suppresses transformation. Mutation of individual mitogen-activating protein kinase phosphoacceptor sites in TCF/Elk-1 also compromises v-raf-activated expression of a Gal-Elk/Gal-chloramphenicol acetyltransferase reporter system. However, in at least one instance the introduction of glutamate, but not aspartate, at a phosphoacceptor site is compatible with activation. These results provide compelling evidence that phosphorylation of TCF/Elk-1 by Erk2 is a major link in the Raf-1 kinase-dependent signal transduction pathway that activates c-fos expression.

Repression of AP-1-stimulated transcription by c-Ets-1.

The transcriptional activities of c-Ets-1 and v-Ets and their functional interaction with the AP-1 factor c-Jun were investigated. Several recombinant Ets proteins were produced and purified either from bacteria or from insect cells. Plasmid DNAs that contained the polyoma virus enhancer Ets/AP-1 element were used as templates for in vitro transcription assays in the presence of HeLa nuclear extract and various combinations of the Jun and Ets proteins. Under these conditions full-length c-Ets-1 on its own does not markedly influence transcription but abolishes the strong transcriptional stimulation normally elicited by Jun. This repression depends on the Ets-binding site and on specific features of c-Ets-1 structure, as both v-Ets and a natural splicing variant c-Ets-1 (delta VII) fail to inhibit Jun activity. These findings suggest that c-Ets may act both as a transcriptional repressor or activator depending on promoter context and splicing pattern.

Transformation of avian fibroblasts overexpressing the c-rel proto-oncogene and a variant of c-rel lacking 40 C-terminal amino acids.

The v-rel oncogene was derived from the c-rel proto-oncogene, which encodes a transcriptional activator. Expression of v-rel transforms avian hematopoietic cells and fibroblasts. Here we report that overexpression (via a replication-competent retroviral vector) of full-length c-Rel as well as a 40-amino-acid, carboxy-terminal deletion construct of c-Rel (c-Rel delta) resulted in the morphological transformation of chicken embryo fibroblasts (CEFs). Subcellular localization of Rel polypeptides in these transformed cells as determined by immunofluorescence and immunoprecipitation revealed their presence in both the nucleus and the cytoplasm, with the majority of Rel polypeptides showing cytoplasmic localization. Cytoplasmic localization could be due to interaction with I kappa B molecules, and in fact, the overexpression of c-Rel or the C-terminal deletion construct of c-Rel resulted in an increase in the levels of mRNA encoding the avian I kappa B protein pp40 and the avian homolog of the NF-kappa B protein, p105. However, expression of v-Rel resulted in the induction of pp40 mRNA only. While c-Rel was a weak activator of kappa B-mediated transcription of a reporter construct in transformed CEFs, v-Rel and c-Rel delta were transcriptional repressors. However, in spite of these differences, all of these proteins resulted in the transformation of CEFs.