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.

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.

Expression of MYB proteins in avian hematopoietic tissues.

MYB gene products are thought to be regulators of cellular replication and of differentiation. The major product of the avian MYB gene is a 75 kDa nuclear phosphoprotein which can activate transcription. A minor 89 kDa MYB protein of unknown function has also been described in murine and human cells. Additional heterogeneity at the level of MYB RNA which could affect the structure of MYB proteins has been described in several species. Such heterogeneity could explain the diverse effects of the MYB gene. To investigate the possible existence of heterogeneous and/or cell lineage-specific MYB proteins, five different avian hematopoietic tissues (bone marrow, bursa of Fabricius, embryonic spleen, thymus and yolk sac) were examined by immunoprecipitation with several MYB-specific antisera and SDS-PAGE analysis. In all five tissues there was a 75 kDa protein of uniform size which varied in abundance in a tissue-specific manner paralleling that observed for the 4.0 kb MYB RNA. A less abundant 89 kDa protein was also detected by several antisera in bone marrow, spleen, thymus and yolk sac but not in bursa. This 89 kDa MYB protein appears to be analogous to the 89 kDa MYB protein encoded by a minor but larger (360 nucleotides) MYB mRNA in murine and human cells. Immunoprecipitation of MYB proteins with an antiserum specific for exons 8 and 9 revealed a 74 kDa protein which co-precipitated and appeared to be complexed with p75 in normal hematopoietic cells and with the 48 kDa product of v-myb in leukemic cells.

erg, an ets-related gene, codes for sequence-specific transcriptional activators.

E26 is a replication-defective avian acute leukemia virus which causes erythroblastosis and myeloblastosis in chickens. It carries two distinct oncogenes, v-myb and v-ets, both of which contribute to its transforming properties. Several genes related to the ets oncogene (c-ets-1, ets-2, erg, elk-1, elk-2, PU.1/Spi-1, E74 and Fli-1) have been described. Previously we have shown that the erg gene (ets-related gene) codes for at least two proteins (erg-1 and erg-2) because of alternative splicing and alternative usage of the initiation codon. We have expressed erg-1 and erg-2 proteins in Escherichia coli and have used these recombinant proteins to show that they bind to DNA in a sequence-specific manner. erg proteins exhibited different sequence specificity and affinity for the oligonucleotides recognized by c-ets-1, ets-2, some of PU.1/Spi-1 and elk-1, suggesting that the DNA-binding specificities of erg and other members may overlap but are not necessarily identical. The erg gene was found to transactivate a reporter gene that was linked to erg target sequences. These results suggest that erg-1 and erg-2 are sequence-specific transcriptional activators like the other members of the ets oncogene superfamily which represent a distinct class of transcriptional activators.

Human ERG-2 protein is a phosphorylated DNA-binding protein--a distinct member of the ets family.

We describe the identification of the ERG-2 gene products using an antibody raised against recombinant human ERG-2 protein. ERG-2 is a nuclear phosphoprotein and binds to purine-rich sequences (C/G)(C/a)GG-AA(G/a)T. ERG-2 protein, with a half-life of 21 h, is considerably more stable than the short-lived ETS-1 or ETS-2 proteins. Its phosphorylation is stimulated by phorbol myristate acetate (PMA), but not by Ca2+ ionophore treatment. ETS-1 protein is phosphorylated by Ca(2+)-dependent events, whereas ERG-2 protein is phosphorylated by activation of protein kinase C, suggesting their involvement in distinct signal transduction mechanisms. The expression of ERG-2 protein is restricted to few cell types and is high in early myeloid cells, indicating that it may function at an early stage of hematopoietic lineage determination. The DNA-binding sequence for ERG-2 protein is identified by using a random oligonucleotide selection procedure. The selected sequence is very similar to the binding sequence determined for human ETS-1 using the same method. Like other ets proteins, ERG-2 is a sequence-specific DNA-binding protein and is expressed at higher levels in early myeloid cells than in mature lymphoid cells. These results suggest that it may act as a regulator of genes required for maintenance and/or differentiation of early hematopoietic cells.

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.

Characterization of v-sis protein expressed in silkworm larvae using the Bombyx mori nuclear polyhedrosis virus vector.

The v-sis oncogene of simian sarcoma virus encodes a protein which is homologous to the human platelet-derived growth factor B-chain. The v-sis protein undergoes a series of processing steps including dimer formation and proteolytic digestion to generate several molecular sizes of the protein. Two of these v-sis proteins were expressed alone or as polyhedrin-sis fusion proteins using the Bombyx mori nuclear polyhedrosis virus vector. The polyhedrin-sis fusion proteins contained a collagenase-sensitive site at the junction. The expression levels of the fusion proteins whose polyhedrin portions consisted of only 8 amino-terminal amino acids were 3-4 times higher than those of non-fusion proteins. One of these fusion proteins was expressed in silkworm larvae and the v-sis protein was isolated from the fusion protein by collagenolysis followed by chromatography. Because the purified v-sis protein exhibited the same molecular size on SDS-polyacrylamide gels under reducing and non-reducing conditions, it was concluded to be monomeric in structure. It possessed chemotactic activity but lacked mitogenic activity. In addition, a small amount (approximately 1%) of monomeric v-sis protein was converted in vitro to the mitogenically active v-sis protein, which could be a homo-dimer.

Large-scale purification of gp70 from Moloney murine leukemia virus.

The external envelope glycoprotein, gp70, of the Moloney murine leukemia virus was extracted from NIH 3T3 cells utilizing the detergent n-octyl-beta-D-glycopyranoside. The extracted gp70 was sequentially purified utilizing lectin-affinity, anion-exchange, and molecular-exclusion chromatography techniques. Approximately 10 mg of gp70 was purified by this method and shown to be 95% homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of purified gp70 from Moloney murine leukemia virus was confirmed by amino acid analysis, amino-terminal sequencing, and immunoreactivity with a monoclonal antibody raised against gp70. The procedure is rapid, utilizes commercially available media, and can be used to purify large amounts of retroviral envelope glycoprotein from virus.

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.

Envelope glycoprotein gp51 of bovine leukemia virus is differently glycosylated in cells of various species and organ origin.

The carbohydrate moiety of the envelope glycoprotein gp51 of bovine leukemia virus, American strain, was studied. The virus was grown in ovine, bovine, porcine, bat and rat cells of various organ specificities. The gp51 was purified by immunoaffinity chromatography from virions of ten different virus-producing cells derived from various body organs of different species. Highly purified glycoproteins (single band in PAGE) were compared for their electrophoretic mobility, for the presence of epitopes by a battery of monoclonal antibodies, and for the glycosylation pattern by lectin blot analysis. Electrophoretic analysis of all tested glycoproteins deglycosylated by glycopeptidase F detected the same polypeptide backbone according to PAGE. The glycoproteins produced in rat cells migrated faster in PAGE, as detected in cells or in virions, than those produced in ovine cells. The pattern of their glycosylation was found to be dependent on the type of cells used for virus production. The differences in glycosylation were most pronounced when comparing the glycoprotein produced in ovine cells versus bat or rat cells. Changes in epitope expression were also detected. The differences in the patterns of glycosylation and in the accessibility of epitopes owing to the virus production in various kind of cells are discussed from virus infectivity and vaccine points of view.

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.

Improvement of simultaneous detection of antibodies to Gag and envelope antigens of human T-lymphotropic virus type I by western immunoblot assay.

To determine seropositivity for human T-lymphotropic virus type I (HTLV-I), we attempted to improve the detection system that uses antibody to HTLV-I Env in Western immunoblotting (WB) by adding an envelope glycoprotein (gp46) purified from the culture fluid of HTLV-I-producing cells by immunoaffinity chromatography and gel chromatography. In this WB, 177 of 179 serum samples showing seropositivity in an indirect immunofluorescence assay showed positive reactions to the gp46 envelope antigen as well as to p19, p24, and p53 Gag antigens. The remaining two samples showed negative reactions to p24. False-positive results were not found for 533 indirect immunofluorescence assay-negative serum samples, although one band to p19 or p24 was observed in 46 of the 533 samples. These 46 samples did not react to p53 and gp46, suggesting that these samples belonged to the indeterminate group in accordance with the criteria proposed by the World Health Organization. Therefore, this improved WB can be used for the confirmation of seropositivity.

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.

Verification of HTLV-I infection in the Solomon Islands by virus isolation and gene amplification.

We report the detection of human T-lymphotropic virus type I (HTLV-I) genomic sequences by polymerase chain reaction in lymphocyte cultures of three unrelated native Solomon Islanders, including a patient with HTLV-I myeloneuropathy, residing in widely separated regions. In addition, we have isolated HTLV-I from T-cell lines derived from two of these individuals. Virus-specific proteins of 15, 19, 24, 46 and 53 kilodaltons were detected by immunofluorescence and Western immunoblot, using serum from a Colombian patient with HTLV-I myeloneuropathy, sera from HTLV-I-infected rabbits, and monoclonal and polyclonal antibodies against HTLV-I gag and env gene products. Amplification of HTLV-I gag, pol and env sequences by polymerase chain reaction confirmed that the viral isolates were HTLV-I, not HTLV-II. Our data clearly demonstrate that HTLV-I does exist in Melanesia. Although the Solomon Islands viral isolates resemble prototype strains of HTLV-I, we believe they represent variants of HTLV-I, particularly in the light of our recent isolation of an HTLV-I variant from Papua New Guinea. Nucleotide sequence analysis of these viral strains, now in progress, should clarify the molecular epidemiology and phylogeny of HTLV-I.

Expression and characterization of kinase-active v-erbB protein using a baculovirus vector system.

The v-erbB gene is an oncogene of the avian erythroblastosis virus encoding a protein that is a truncated version of the epidermal growth factor receptor. The v-erbB protein was expressed alone or as polyhedrin-erbB fusion proteins using the Bombyx mori nuclear polyhedrosis virus vector. The expression level of the fusion protein whose polyhedrin portion consisted of only 8 amino-terminal amino acids was more than ten times higher than that of the non-fusion protein. Studies with tunicamycin showed that the recombinant v-erbB proteins were glycosylated. The recombinant protein autophosphorylated tyrosine residues, and phosphorylated a synthetic tyrosine-containing peptide and lipocortin I. These observations indicate that functional v-erbB protein can be expressed in silkworm-derived cells, and furthermore, that this system can be used for large-scale production.

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.