Cooperation between bovine leukaemia virus transactivator protein and Ha-ras oncogene product in cellular transformation.

Human T-lymphotropic viruses (HTLV-I and -II) and bovine leukaemia virus (BLV) express transactivator proteins able to increase long terminal repeat (LTR) directed viral expression. These transacting factors are though to be involved in the induction of leukaemia by these viruses. Transfection of BLV transactivator p34tax together with Ha-ras immortalizes and transforms rat embryo fibroblasts, in vitro. The transformed cell induce tumours in nude mice. These data emphasize the causal role exerted by p34tax in in vivo tumorigenesis.     

Activator-dependent and activator-independent defective recombinant retroviruses from bovine leukemia virus.

The replication-competent bovine leukemia virus (BLV) has been modified for use as a vector for foreign genes. The gag, pol, env, and pX regions of the virus were replaced by an exogenous nuclear location signal LacZ (nlsLacZ) or SVnlsLacZ gene. Transfection of the ovine cell line FLK-BLV, which expresses all BLV proteins from a wild-type provirus, with this viral DNA resulted in a viral titer of 10(4) CFU/ml. The inclusion of a large portion of the gag region did not significantly increase the titer. Both activator-dependent and activator-independent retroviruses were constructed. In activator-dependent vectors, the expression of the insert was dependent on the presence of the Tax protein, which activated the BLV long terminal repeat. In activator-independent vectors, the expression of the insert was constitutive because of the presence of an internal promoter. Infections with the recombinant retrovirus were inhibited by specific neutralizing antibodies. The structure of the transduced genetic material was not rearranged. BLV vectors encoding a reporter nlsLacZ gene, the product of which can be detected in single cells, greatly simplified studies of their biological properties. Determination of the host range of BLV vectors established that BLV-based recombinant retroviruses are effective in the transduction of genes in a variety of species and cell types.     

Dileucine and YXXL motifs in the cytoplasmic tail of the bovine leukemia virus transmembrane envelope protein affect protein expression on the cell surface

Several retroviruses downmodulate the cell surface expression of envelope (Env) proteins through peptide sequences located in the cytoplasmic tail of the transmembrane (TM) subunit. We investigated whether cell surface expression of a chimeric protein containing the cytoplasmic domain of the TM protein (CTM) of bovine leukemia virus (BLV) was regulated by two membrane-proximal dileucine motifs or by tyrosine Y487 or Y498 in YXXL motifs. A chimeric protein composed of the extracellular and membrane-spanning portions of human CD8-alpha plus a wild-type (wt) BLV CTM was detectable on the surface of only 40% of the cells in which it was transiently expressed. Replacement of either dileucine pair with alanines increased the level of surface display of chimeric proteins. Nearly all cells became surface positive when both dileucine motifs were altered simultaneously and when either an N-terminal segment containing both dileucine motifs or a C-terminal segment containing all YXXL motifs was deleted. In contrast, replacement of Y487 or Y498 with alanine or phenylalanine enabled only small increases in surface display compared with wt levels. Chimeric proteins had similar stabilities but were downmodulated from the cell surface at three different rates. Point mutants segregated into each of the three groups of proteins categorized according to these different rates. Interestingly, Y487 mutants were downmodulated less efficiently than Y498 mutants, which behaved like wt. CD8-CTM chimeric proteins were phosphorylated on serine residues, but the native BLV Env protein was not phosphorylated either in transfected cells or in a lymphoid cell line constitutively producing BLV. Thus, both dileucine and YXXL motifs within the BLV CTM contribute to downmodulation of a protein containing this domain. Interactions with other proteins may influence surface exposure of Env protein complexes in virus-infected cells, assisting in viral evasion of adaptive immunity.     

A cyclic AMP-responsive DNA-binding protein (CREB2) is a cellular transactivator of the bovine leukemia virus long terminal repeat.

To gain insight into the cellular regulation of bovine leukemia virus (BLV) trans activation, a lambda-gt11 cDNA library was constructed with mRNA isolated from a BLV-induced tumor and the recombinant proteins were screened with an oligonucleotide corresponding to the tax activation-responsive element (TAR). Two clones (called TAR-binding protein) were isolated from 750,000 lambda-gt11 plaques. The binding specificity was confirmed by Southwestern (DNA-protein) and gel retardation assays. Nucleotide sequence analysis revealed that TAR-binding protein is very similar to the CREB2 protein. It contains a leucine zipper structure required for dimerization, a basic amino acid domain, and multiple potential phosphorylation sites. A vector expressing CREB2 was transfected into D17 osteosarcoma cells. In the absence of the tax transactivator, the CREB2 protein and the cyclic AMP-dependent protein kinase A activate the BLV long terminal repeat at a basal expression level: trans activation reached 10% of the values obtained in the presence of tax alone. These data demonstrate that CREB2 is a cellular factor able to induce BLV long terminal repeat expression in the absence of tax protein and could thus be involved in the early stages of viral infection. In addition, we observed that in vitro tax-induced trans activation can be activated or inhibited by CREB2 depending on the presence or absence of protein kinase A. These data suggest that the cyclic AMP pathway plays a role in the regulation of viral expression in BLV-infected animals.     

Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model

Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV gag, pol, and env and related cis-acting sequences but lacks tax, rex, RIII, and GIV and most of the BLV long terminal repeat sequences, including the cis-acting Tax and Rex response elements. The novel SGV virus is replication competent and replicates a selectable vector to a titer similar to that of the parental BLV in cell culture. The overall goal of this study was to test the hypothesis that infection with BLV SGV is nonpathogenic in rabbits. BLV infection of rabbits by inoculation of cell-free BLV or cell-associated BLV typically causes an immunodeficiency-like syndrome and death by 1 year postinfection. We sought to evaluate whether in vivo transfection of BLV provirus recapitulates pathogenic BLV infection and to compare BLV and BLV SGV with respect to infection, immunogenicity, and clinical outcome. Three groups of rabbits were subjected to in vivo transfection with BLV, BLV SGV, or negative control DNA. The results of our 20-month study indicate that in vivo transfection of rabbits with BLV recapitulates the fatal BLV infection produced by cell-free or cell-associated BLV. The BLV-infected rabbits exhibited sudden onset of clinical decline and immunodeficiency-like symptoms that culminated in death. BLV and BLV SGV infected peripheral blood mononuclear cells and induced similar levels of seroconversion to BLV structural proteins. However, BLV SGV exhibited a reduced proviral load and did not trigger the immunodeficiency-like syndrome. These results are consistent with the hypothesis that BLV SGV is infectious and immunogenic and lacks BLV pathogenicity in rabbits, and they support the use of this modified proviral vector delivery system for vaccines against complex retroviruses like BLV.     

Involvement of bovine leukemia virus in induction and inhibition of apoptosis

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G, that has an enhanced capacity to transactivate the long terminal repeat (LTR) of BLV and the cellular proto-oncogene c-fos when compared with wild-type Tax (wt-Tax). We demonstrate here that an infectious strain of BLV containing the mutant D247G form of Tax also differs in its capacity to modulate cell survival both positively and negatively. When peripheral blood mononuclear cells (PBMCs) infected with wild-type or mutant BLV are cultured ex vivo with staurosporine, an agent known to induce a mitochondrial caspase cascade pathway regulating apoptosis, the rate of apoptosis is reduced to a greater extent in cells infected with mutant BLV than wild-type BLV, consistent with previous observations in cultures without staurosporine. The increase in survival was associated with an increase in expression of mRNA of bcl-xl but not bcl-2 and bax ex vivo. In contrast, when a tissue culture-adapted cell line, 293T, was transiently transfected with either wild-type or mutant BLV, apoptosis was induced. The increase in the rate of apoptosis was higher in cells transfected with mutant BLV. The same difference was noted in cells transiently transfected with wild-type and mutant D247G Tax, suggesting that the observed positive and negative modulation of cell survival is attributed to the functional characteristics of mutant D247G Tax.     

Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity.

A recombinant bovine leukemia virus (BLV) was constructed in which the X region was replaced with the bacterial neomycin resistance gene controlled by the simian virus 40 early promoter. This virus, termed BLV-SVNEO, is a self-packaging, activator-dependent retroviral vector. Introduction of the plasmid pBLV-SVNEO into mammalian cells resulted in constitutive expression of the neo gene, whereas the BLV structural genes, gag, pol, and env, were expressed only in the presence of the two regulatory proteins, Tax and Rex. The production and release of recombinant virus by cells transfected with pBLV-SVNEO were proportional to the number of G418-resistant colonies that developed after susceptible cells were exposed to the filtered culture medium. BLV-SVNEO was able to infect cell lines of human, bovine, canine, feline, and murine origin. BLV-producing cell lines were resistant to superinfection with BLV-SVNEO. This cell-virus system should facilitate molecular genetic studies of BLV and will provide a rapid, quantitative measure of BLV infectivity in a variety of cell types. These studies also demonstrate the feasibility of using activator-dependent retroviral vectors such as BLV-SVNEO to deliver foreign genes into cells and eventually animals.     

A mutant form of the tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity,increases expression and propagation of BLV in vitro but not in vivo

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.     

JDV与三种牛反转录病毒相互关系的初步研究

为研究JDV与其它三种牛反转录病毒BIV、BLV、BFV的相互作用关系,将以JDV、BIV、BLV、BFV的LTR为启动子,以Luc为报告基因的质粒和以上病毒反式激活因子的表达质粒共转染BLl2细胞系,通过瞬时表达分析试验证明了JDV和BIV的LTR和Tat之间亲缘关系很近,能够相互激活;JDV Tat可以反式激活BLVLTR,BLVTax不能激活JDVLTR;JDVLTR上存在BFVTas的应答元件;BLV、BFV和BIV的LTR和反式激活因子问不存在相互激活。     

Growth inhibition of cancer cells by co-transfection of diphtheria toxin A-chain gene plasmid with bovine leukemia virus-tax expression vector

We constructed a plasmid containing bovine leukemia virus (BLV)-tax gene driven by SR alpha promoter, designated as pME-BLVtax, to activate the promoter of the long terminal repeat (LTR) of BLV in various tumor cells. Activation of the promoter of BLV-LTR by pME-BLVtax was confirmed by luciferase assay. When the cells, such as COS-1, C8, and KU-1, were transfected with a plasmid pBLV-LUC1, which contained the luciferase gene under the control of BLV-LTR, and pME-BLVtax, luciferase was expressed in these cells, whereas no luciferase gene expression was observed when only pBLV-LUC1 was introduced into the cells. Activation of the BLV-LTR promoter was regulated by pME-BLVtax and 0.5 microg of pME-BLVtax was sufficient for the expression of the gene under the control of BLV-LTR. Furthermore, pME-BLVtax was used to direct the cell expression of the gene for diphtheria toxin A-chain under the control of BLV-LTR (pLTR-DT) to various tumor cell lines, KU-1, C8, COS-1, BL2M3, and HeLa cells. The transfection was carried out with cationic liposomes. In this experiment, co-transfection of pLTR-DT with pME-BLVtax exerted selective growth inhibitory effects on the tumor cell lines. Moreover, three co-introductions of pLTR-DT with pME-BLVtax into the cell lines resulted in significant inhibition of the cell growth. This result suggests that the delivery of the pLTR-DT and pME-BLVtax genes into tumor cells by the use of cationic liposomes may be potentially useful as a novel approach for the treatment of tumor cells.     

Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes.

Recombinant retroviruses containing the trans activator genes of human T-cell leukemia virus (HTLV) type II and human T-cell lymphotropic virus type III were constructed. The trans activator genes tat II and tat III were inserted into the murine retroviral vector pZIPNEOSV(X)1. Recombinant plasmids were transfected into the psi 2 and psi AM packaging cell lines that produce murine leukemia virions containing no retroviral RNA. Functional tat II and tat III gene products were expressed as demonstrated by trans activation of HTLV type I and II and human T-cell lymphotropic virus type III long terminal repeat-directed gene expression in the respective infected cells. Use of these recombinant vectors permits high-efficiency gene transfer into a wide variety of cells, thereby providing the opportunity to study the biochemical effects associated with tat II and tat III gene expression.     

Two retroviruses packaged in one cell line can combined inhibit the replication of HIV-1 in TZM-bl cells

The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids’ functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3T0V, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bl cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.     

A gas-inducible expression system in HEK.EBNA cells applied to controlled proliferation studies by expression of p27(Kip1)

We describe an efficient inducible gene expression system in HEK.EBNA cells, a well-established cell system for the rapid transient expression of research-tool proteins. The transgene control system of choice is the novel acetaldehyde-inducible regulation (AIR) technology, which has been shown to modulate transgene levels following exposure of cells to acetaldehyde. For application in HEK.EBNA cells, AlcR transactivator plasmids were constructed and co-expressed with the secreted alkaline phosphatase (SEAP) gene under the control of a chimeric mammalian promoter (P(AIR)) for acetaldehyde-regulated expression. Several highly inducible transactivator cell lines were established. Adjustable transgene induction by gaseous acetaldehyde led to high induction levels and tight repression in transient expression trials and in stably transfected HEK.EBNA cell lines. Thus, the AIR technology can be used for inducible expression of any desired recombinant protein in HEK.EBNA cells. A possible application for inducible gene expression is a controlled proliferation strategy. Clonal HEK.EBNA cell lines, expressing the fungal transactivator protein AlcR, were engineered for gas-adjustable expression of the cell-cycle regulator p27(Kip1). We show that expression of p27(Kip1) via transient or stable transfection led to a G1-phase specific growth arrest of HEK.EBNA cells. Furthermore, production pools engineered for gas-adjustable expression of p27(Kip1) and constitutive expression of SEAP showed enhanced productive capacity.     

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor.

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.