Characterization of R peptide of murine leukemia virus envelope glycoproteins in syncytium formation and entry.

The C-terminal R peptide of ecotropic murine leukemia virus (MLV) envelope protein (Env) negatively controls membrane fusion activity. The R peptide cleavage during virion maturation activates its fusogenicity and is required for viral entry. We analyzed fusogenicity and transduction efficiency of mutant Env proteins of ecotropic, amphotropic, polytropic, and xenotropic MLVs. As the result, we found that the hydrophobic amino acid residues around the R peptide cleavage site are important for membrane fusion inhibition by the R peptide. In addition, we found that Env complexes with R peptide-truncated and -containing Env proteins have lower fusogenicity and transduction efficiency than those with the R-peptide-truncated Env alone, suggesting that efficient R peptide cleavage is required for efficient MLV vector transduction. The role of R peptide cleavage in amphotropic, polytropic, and xenotropic MLV infection has not been investigated. We found in this study that the R peptide cleavage is required for amphotropic, xenotropic, and polytropic MLV vector transduction, like with ecotropic MLV. The R-peptide-truncated Env proteins of the xenotropic and polytropic MLVs, however, had much lower fusogenicity than those of the ecotropic and amphotropic MLVs. These results provide valuable information for construction of efficient MLV vectors and for understanding the retroviral entry mechanism.

Genistein, a protein tyrosine kinase inhibitor, suppresses the fusogenicity of Moloney murine leukemia virus envelope protein in XC cells.

XC cells are highly susceptible to syncytium formation by infection of ecotropic murine leukemia viruses (MLVs) and by expression of their envelope protein (Env). By this property, XC cells are widely used to determine titers of ecotropic MLVs. Number of plaques resulted from the syncytium formation in XC cells by ecotropic MLV infection is corresponding to number of the viral particles. XC cells had been established from a v-src-induced rat tumor. It has been reported that transformed cells are more sensitive to Mo-MLV-induced syncytium formation than non-transformed cells. To assess whether the transformation by v-src oncogene in XC cells is involved in the high sensitivity to ecotropic MLV-induced syncytium formation, XC cells were treated with genistein, a protein tyrosine kinase inhibitor. Genistein suppressed the syncytium formation between XC cells and ecotropic Env-expressing 293T cells. This result indicates that protein tyrosine kinase activity is associated with the high sensitivity of XC cells to ecotropic Env-induced syncytium formation.

A hyperthermostable bacterial histone-like protein as an efficient mediator for transfection of eukaryotic cells.

Gene delivery has shown potential in a variety of applications, including basic research, therapies for inborn genetic defects, cancer, AIDS, tissue engineering, and vaccination. Most available systems have serious drawbacks, such as safety hazards, inefficiency under in vivo-like conditions, and expensive production. When using naked DNA, for instance, a large amount of ultrapure DNA has to be applied as a result of degradation by nucleases. Similarly, the use of eukaryotic histones, synthetic peptides, or peptide nucleic acids may be limited by high production costs. We have demonstrated a biotechnologically feasible and economical approach for gene delivery using the histone-like protein from the hyperthermostable eubacterium Thermotoga maritima, TmHU as an efficient gene transfer reagent. HU can be easily isolated from recombinant Escherichia coli, is extraordinarily stable, and protects dsDNA from thermal denaturation. This study demonstrates its use as an inexpensive tool for gene delivery.

Transgenic zebrafish for detecting mutations caused by compounds in aquatic environments.

We have established a transgenic zebrafish line carrying a shuttle vector plasmid (pML4) for detecting mutagens in aquatic environments. The plasmid contains the rpsL gene of Escherichia coli as a mutational target gene, and the kanamycin-resistance gene for recovering the plasmid from the chromosomal DNA. To evaluate the system, we treated embryos of the transgenic fish with N-ethyl-N-nitrosourea (ENU), which induces a dose-dependent increase in the mutation frequency of the target gene. The mutation spectrum was consistent with the proposed mechanism of ENU mutagenesis. Similarly, treating the embryos with benzo[a]pyrene or 2-amino-3, 8-dimethylimidazo[4,5- f]quinoxaline, which are found in naturally polluted water, significantly increased the frequency of mutations in the target gene.

Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.

We have developed a method for high-efficiency window separation of cDNA display by increasing the specificity of priming in reverse transcription. In the conventional method, two-base anchored oligo(dT) primers (5'dT16VN3', where N is any base and V is G, A or C) are used to make windows for the display of transcripts. However, reverse transcriptase often extends misprimed oligonucleotides. To avoid mispriming from dT16VN primers, we have developed two new technologies. One is higher temperature priming with reverse transcriptase thermoactivated by the disaccharide trehalose. The other is the use of competitive oligonucleotide blockers that hybridize to the non-selectively primed mRNAs, preventing the mispriming from the VN site. These methods were combined to improve restriction landmark cDNA scanning (RLCS), resulting in the elimination of the redundant signals that appear in different windows. This was achieved by the increased specificity of initiation of reverse trans-cription from the beginning of poly(A) sites. This method paves the way for the precise visualization of transcripts to allow expression profiles in individual tissues and at each developmental stage to be understood.

Impaired activation and binding of the erythropoietin receptor by a mutant gp55 of Friend spleen focus-forming virus, which has a cytoplasmic domain.

A murine erythroleukemogenic retrovirus, Friend spleen focus-forming virus, encodes an envelope protein-like membrane glycoprotein (gp55) in its defective env gene which is responsible for activation of the erythropoietin receptor (EpoR) and the abnormally rapid proliferation of erythroid precursor cells. The S34 mutant gp55, which possesses an additional cytoplasmic domain, is nonpathogenic and its processing to the cell surface is severely reduced compared to that of the wild-type gp55. In this study, we found that the S34 mutant gp55 neither binds to nor activates the EpoR. The S34 mutant gp55 formed disulfide-bonded homodimers in the rough endoplasmic reticulum (RER) membrane much less efficiently than the wild-type gp55, which is consistent with the proposal that homodimer formation is a prerequisite for gp55 to be exported from the RER. We found that the wild-type gp55 that is bound to EpoR in the RER consists of a large number of monomers and a small number of dimers, suggesting that monomers of the S34 mutant gp55 have lost the ability to bind to the EpoR. The 1-bp insertion present in the wild-type gp55 gene, causing a loss of the cytoplasmic domain, is essential for pathogenicity in that it renders the encoded protein capable of both binding to the EpoR and transport to the cell surface.

Pathogenicity of a mutant friend spleen focus-forming virus encoding an Env-like membrane glycoprotein (gp55) with substitution by a xenotropic murine leukemia virus Env gp70 sequence.

Friend spleen focus-forming virus (F-SFFV) is a replication-defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)-like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus-inducing (MCF) murine leukemia virus (MuLV) Env gp70-derived sequence in its amino-terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino-terminal region. XE gp55 displayed almost the same biological activities as the wild-type gp55, excluding the above possibility.

Sequence flexibility in the polytropic env gp70-derived region of the membrane glycoprotein (gp55) of Friend spleen focus-forming virus affects its biological activity.

We previously reported (N. Watanabe, M. Nishi, Y. Ikawa, and H. Amanuma, J. Virol. 65:132-137, 1991) that the mutant Friend spleen focus-forming virus (F-SFFV(MS)), which encodes a mutant gp55 membrane glycoprotein with an ecotropic env gp70 sequence, was nonpathogenic. Here we injected the F-SFFV(MS)-Friend murine leukemia virus (F-MuLV) clone 57 complex into newborn DBA/2 mice. We obtained four groups of pathogenic variant F-SFFV complexes, each showing a different degree of pathogenicity in adult mice and a different gp55 profile. Of these, group 1 variant F-SFFV was particularly interesting, because it was the most frequently obtained and because it produced doublet bands of gp55 (59 and 57 kDa), neither of which reacted with the nonecotropic gp70-specific monoclonal antibody, and because its DNA intermediate did not hybridize with the nonecotropic env-specific probe. Cloning and DNA sequence analysis of the env region of one isolate of the group 1 variant F-SFFV revealed that this virus consisted of two distinct F-SFFV genomes; one (clone 117) differed from the other (clone 118) due to the presence of a 39-bp in-frame deletion. Reconstitution to full-length F-SFFV genomes and a pathogenicity assay showed that each reconstituted F-SFFV was pathogenic, with clone 117 showing a higher degree of pathogenicity than clone 118. Both reconstituted F-SFFVs caused activation of the mouse erythropoietin receptor in the factor-independent cell proliferation assay, although much less efficiently than the wild-type polycythemia-inducing isolate F-SFFVp. Clone 118 produced a gp55 of 59 kDa, while clone 117 produced one of 57 kDa. Clone 118 had a substitution by the F-MuLV clone 57 gp70 sequence, indicating that it was derived from the F-SFFV(MS) env gene by a homologous recombination with the F-MuLV clone 57 env gene. The site of the 39-bp deletion in clone 117 corresponded to the portion of the clone 118 sequence which was unique to the ecotropic env genes. These results indicated the importance for the biological activity of gp55 of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.

Inhibition of gene expression from the human c-erbB gene promoter by a retroviral vector expressing anti-gene RNA.

Anti-gene is a potent inhibitor of transcriptional promoter activity and subsequent gene expression. This property has been exploited to suppress the expression of a variety of oncogenes for regulating tumor proliferation or viral activities. In this paper, we describe a novel retroviral vector designed to express human c-erbB anti-gene RNA and to reduce the promoter activity in the cells. Mouse fibroblast NIH3T3 cells were stably transfected with an expression construct containing a truncated human c-erbB gene promoter fused to the firefly luciferase reporter gene. Infection into these cells of the c-erbB anti-gene retroviral vector targeted to the 26 bp pyrimidine-rich element in the human c-erbB gene promoter resulted in a dose-dependent decrease in the luciferase activity of the cells. Retroviral vector expressing anti-gene RNA may be useful as an alternative program of gene regulation in the cells.

Mutational analysis of the structure-function relationship of the leukemogenic membrane glycoprotein (GP55) of Friend spleen focus-forming virus (F-SFFV).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in adult mice. F-SFFV encodes an envelope protein-like membrane glycoprotein called gp55 in its defective env gene. Gp55 is responsible for the early stage of leukemogenesis by F-SFFV by specifically binding to and activating the murine erythropoietin receptor (EPO-R). Gp55 has a polytropic env sequence in its N-terminal portion. This portion probably contains the binding site for the EPO-R. In order to obtain a clue for the structure of the binding site to the EPO-R, we isolated and analyzed many spontaneous revertant F-SFFVs which derived from the non-leukemogenic mutant F-SFFV having an ecotropic env sequence instead of the polytropic env sequence in its gp55 gene.

The role of tumor-associated antigen in bovine leukemia virus-induced lymphosarcoma.

Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. To clarify the way in which BLV-infected cattle progress from the asymptomatic stage to the lymphoma stage, we produced a monoclonal antibody (MAb) c143 which recognized a tumor-associated antigen (TAA) that is phosphorylated in the transformed state of BLV-infected B-lymphoid cells. Since the nature of c143 TAA was likely to be that of the major histocompatibility complex (MHC) class II antigens, we isolated cDNAs for bovine MHC (BoLA) class II a-chains and b-chains, produced transfectants that expressed a single type of BoLA class II molecules and analyzed them by flow cytometry with c143 MAb. The c143 MAb recognized the transfectant expressing BoLA-DR but not BoLA-DQ. However, the treatment of lymphocytes with c143 or anti-BoLA-DR MAb induced different effects. Although mixed lymphocyte reaction (MLR) was inhibited by the addition of anti-BoLA-DR MAb, the c143 MAb did not inhibit a proliferative response of T cells in MLR. Increased spontaneous proliferation of lymphocytes in healthy donors was obtained in the presence of c143 MAb but not anti-BoLA-DR MAb, and was much in lymphocytes from the carrier. Moreover, the patterns of immunohistological staining for c143 MAb in BLV-infected sheep showed distinguishing differences from those of anti-BoLA-DR MAbs.

Peptide-based bovine leukemia virus (BLV) vaccine that induces BLV-Env specific Th-1 type immunity.

In controlling retrovirus infection and replication, cell-mediated immunity (CMI) is considered to be effective. To develop a synthetic peptide vaccine capable of inducing CMI, mannan-coated liposome encapsulating 20-mer synthetic peptide, spanning the 98-117 amino acids of bovine leukemia virus (BLV) envelope glycoprotein (Env) gp51 was constructed and inoculated to BALB/c mice. The liposome induced specific delayed-type hypersensitivity, lymphocyte proliferative responses, and a weak cytotoxic lymphocyte response. The spleen cells from the immunized mice produced a large amount of IFN-gamma and IL-2, whereas they released neither IL-4 or IL-10. Mannan-coated liposome containing two kinds of peptides (the 121-140 and 142-161 regions of BLV Env gp51) also induced peptide-specific lymphocyte proliferative response and IFN-g production in C57BL/6 mice. Thus, the synthetic T-cell epitope peptide-liposome system augmented a strong Th-1 type immunity in mice.

An effective peptide vaccine to eliminate bovine leukaemia virus (BLV) infected cells in carrier sheep.

Protective effects of the gp51 of bovine leukaemia virus (BLV) expressed by a recombinant baculovirus (rgp51) and synthetic multiple antigenic peptides (MAP) of T-helper, T-cytotoxic, and B-cell epitopes of gp51 were investigated against BLV challenge. Two and three sheep were immunized with rgp51 and a mixture of peptides with Freund's complete adjuvant, respectively. BLV was detected from all the immunized sheep at 2 weeks and showed peak levels at 4 weeks after the challenge. However, in two sheep immunized with the mixed peptides, the titer of BLV gradually decreased and one sheep eliminated BLV completely at 28 weeks after the challenge. These two sheep showed higher lymphocyte proliferative responses against the immunized peptides than the other sheep. One of the sheep also showed the specific cytotoxic lymphocyte activity against the BLV gp51-expressing target in vitro. These results suggest the possibility of the peptide vaccine for elimination of BLV in carrier animals in vivo.

Induction of bovine leukaemia virus Env-specific Th-1 type immunity in mice by vaccination with short synthesized peptide-liposome.

Cell-mediated immunity (CMI) is considered to be effective in controlling retrovirus infection and replication. To develop a peptide-based vaccine capable of inducing CMI, mannan-coated liposome encapsulating 20-mer synthetic peptide, spanning the 98-117 amino acids of bovine leukaemia virus (BLV) envelope glycoprotein (Env) gp51 was constructed. The liposome induced specific delayed-type hypersensitivity, lymphocyte proliferative responses and a weak cytotoxic lymphocyte response in mice. By stimulation with the peptide and BLV virion, the spleen cells from the immunized mice produced a large amount of IFN-gamma and IL-2, whereas they released neither IL-4 or IL-10. These results indicate the augmentation of Th-1 type immunity in mice by the T-cell epitope synthetic peptide-liposome.

Specification of posterior midbrain region in zebrafish neuroepithelium.

BACKGROUND: The developing vertebrate nervous system displays a pronounced anterior-posterior (A-P) pattern, but the mechanism that generates this pattern is poorly understood. We examined through cell-transplantation experiments, when and how the cells in the zebrafish posterior midbrain acquire regional specificity along the A-P axis as shown by pax[b] gene expression. RESULTS: Labelled donor cells from the presumptive midbrain region at various stages were transplanted into more anterior part of unlabelled host embryos of the same developmental stage, and the expression of pax[b] in the donor cells were examined by in situ hybridization. The results indicated that, in the cells from the presumptive midbrain region, expression of pax[b] was determined as early as the 55%-epiboly (6.5 h, early gastrulation) when the underlying hypoblastic layer reached the presumptive midbrain region. We also found that when transplanted heterotopically, anterior, but not posterior, hypoblast cells induced expression of pax[b] in the overlying ectoderm. CONCLUSIONS: Expression of a midbrain specific gene is determined during early gastrulation and the hypoblastic layer plays an important role in this determination process.

Both the changes of six amino acids and the C-terminal truncation caused by a one-base insertion in the defective env gene of Friend spleen focus-forming virus significantly affect the pathogenic activity of the encoded leukemogenic membrane glycoprotein (gp55).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in mice and encodes in its defective env gene an Env-like membrane glycoprotein (gp55). The F-SFFV env gene has three characteristic structures compared with that of ecotropic murine leukemia viruses (MuLVs): substitution by the polytropic MuLV env sequence, a 585-bp deletion, and a 1-bp insertion. All of these characteristic structures are essential for the leukemogenic potential of gp55 of polycythemia-inducing isolates of F-SFFV (F-SFFVp). The 1-bp insertion causes changes of six amino acids and truncation by 34 amino acids at the C terminus. In this study, we constructed 12 mutant F-SFFV genomes starting from the wild-type F-SFFVp and examined the effect of the C-terminal truncation and the six altered amino acids on the pathogenic activity of gp55. The results indicated that at least 18 to 24 amino acids must be deleted from the C terminus for the env product to be pathogenically active. We also found that the six altered amino acids contributed significantly to the pathogenic activity of gp55. Analyses of the cellular processing of these mutant gp55s supported a correlation between the pathogenic activity of gp55 and its efficiency in overall cellular processing.

Midkine enhances fibrinolytic activity of bovine endothelial cells.

A hitherto unknown function of midkine (MK) was found in the regulation of fibrinolytic activity of vascular endothelial cells. Recombinant murine MK enhanced plasminogen activator (PA)/plasmin levels in bovine aortic endothelial cells (BAECs) in a dose- and time-dependent manner. After incubation with 10 ng/ml MK for 18 h, PA and plasmin levels increased 6- and 4-fold, respectively. This effect was attributed to a moderate upregulation of urokinase-type PA expression as well as to a significant down-regulation of PA inhibitor-1 (PAI-1) expression. BAECs constitutively synthesized and secreted MK and its production was enhanced 2-fold with 1 microM retinoic acid or 10 microM retinol. It was found that MK served as a substrate for tissue transglutaminase. In the culture medium, MK existed as a transglutaminase-mediated complex of 36 kDa. Addition of anti-MK antibody to BAEC cultures resulted in a decrease of basal PA activity and an increase of basal PAI-1 levels and attenuated the ability of retinol to enhance PA activity 50% and potentiated the ability to increase PAI-1 levels 4-fold. Furthermore, MK and basic fibroblast growth factor (bFGF) acted more than additively in enhancing PA levels. We conclude that in BAECs MK is a novel autocrine factor sustaining the fibrinolytic property. MK functions as a mediator of retinoid and cooperates with bFGF to enhance fibrinolytic activity of BAECs.

Lipopolysaccharide inhibits activation of latent transforming growth factor-beta in bovine endothelial cells.

The activation of latent transforming growth factor-beta (TGF-beta) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-beta levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-beta activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 microM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-beta secreted into the culture medium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-beta expression as well as the suppression of surface activation of latent TGF-beta. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-beta did not occur in cells maintained in LPS-contaminated culture medium.

Synthetic peptides derived from midkine enhance plasminogen activator activity in bovine aortic endothelial cells.

Chemically synthesized human midkine enhanced plasminogen activator activity and decreased its inhibitor levels in bovine aortric endothelial cells. These activities were preserved in the C-terminal half, but not in the N-terminal half of the midkine molecule. Furthermore, a synthetic peptide of 43 amino acids designated as "C-domain", which formed the compact structure held by two disulfide bonds in the C-terminal half, mimicked intact midkine. Chemically synthesized C-domain of pleiotrophin (43 amino acids), which was 53% identical to midkine C-domain in amino acid sequence, expressed the similar activities. These 43 amino acid peptides are, so far, the shortest peptide able to enhance the fibrinolytic activities of the endothelial cells.