The bovine leukemia virus p34 is a transactivator protein.

Recombinant Moloney murine retroviruses containing the BLV post-envelope long open reading frame were constructed and transfected into the psi 2 packaging cell line. They were shown to encode and to express a 34-kd protein able to transactivate the BLV long terminal repeat-directed gene expression in the respective transfected cells. These data demonstrate that the BLV X-LOR gene encodes a p34 transactivator product. Furthermore, the different cell lines produced infectious recombinant retroviruses capable of transferring X-LOR genes into recipient cells. The availability of the BLV transactivator protein should allow us to understand the role of the transactivator protein in BLV-induced leukemogenesis.     

Expression of bovine leukemia virus protein p24 in Escherichia coli and its use in the immunoblotting assay.

The gag gene encoded protein, p24 of bovine leukemia virus (BLV), was cloned and expressed as thioredoxin-6xHis-p24 protein in Escherichia coli. The bacterial cells carrying plasmid pT7THis-p24 expressed the protein of 38 kDa that was detected by immunoblotting analysis using anti-p24 monoclonal antibodies and sera from BLV infected cattle and sheep. The purified p24 fusion protein was shown to be sensitive and specific for detection of BLV antibodies in the infected cattle.     

Isolation, purification and immunologic specificity of monoclonal antibodies to antigen p24 of bovine leukosis virus]

The aim of the work is to develop optimal conditions for identification and purification of monoclonal antibodies specific to protein p24 of bovine leukemia virus (p24-BLV). Two schemes of isolation and purification of monoclonal antibodies are compared. Purified monoclonal antibodies contain two subunits with molecular weight of about 22,000 and 50,000 Da as determined by sodium dodecylsulphate polyacrylamide gel electrophoresis. They react with p24-BLV in the immunoblot assay.     

Effects of hydrogen peroxide on bovine leukemia virus expression

Several activators of bovine leukemia virus (BLV) expression, including lipopolysaccharides, phorbol esters and calcium ionophores, are known to generate reactive oxygen species (ROS). Therefore the influence of H2O2 on BLV expression in two BLV producing cell lines was investigated. The effect of H2O2 on BLV expression is apparently dose-dependent. Incubation of FLK/BLV cells with low concentrations of H2O2 (2.5 to 10 microM) induced a marked enhancement of BLV p24 synthesis and an activation of the long terminal repeat (LTR). Higher concentrations resulted in a decrease of proliferation, induction of apoptosis and in a decrease of BLV synthesis. Furthermore, in both cell lines H2O2 treatment led to the activation of NF-kappaB. Pretreatment of cells with antioxidants abrogated the H2O2-induced BLV expression. Taken together, our findings suggest that oxidative stress stimulates BLV expression via activation of NF-kappaB, raising the possibility that biological sources of H2O2, such as stimulated phagocytes, may influence BLV expression.     

Cellular pathways involved in the ex vivo expression of bovine leukemia virus.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression.     

Structure of a defective provirus of bovine leukemia virus

Defective proviruses of bovine leukemia virus (BLV) in the genomes of infected cells were investigated by using Southern blotting hybridization analysis with various portions of a cloned BLV DNA as probes. When nine independent tumors of enzootic bovine leukosis with a single proviral copy per cell were examined, a single defective provirus of BLV was found in one tumor and also in a bovine B cell line derived from this tumor. Hybridization analysis of this defective provirus revealed that it underwent deletion between the pol and env genes and contained no major deletion in the other regions.     

Involvement of intracellular Ca2+ in the regulation of bovine leukemia virus expression

The calcium ionophore A23187, which was used to increase the intracellular calcium concentration ([Ca2+]i), was analyzed for effects on bovine leukemia virus (BLV) expression in two BLV infected cell lines. To clarify the role of intracellular free calcium in this response, [Ca2+]i was measured during ionophore treatment with the fluorescent calcium indicator Fura-2. Elevation of intracellular calcium under these conditions caused an enhancement of BLV gp51 and p24 synthesis as well as an activation of the BLV long terminal repeat (LTR) in a dose-dependent manner. Furthermore, it was observed that elevated levels of intracellular calcium following A23187 stimulation lead to activation of NF-kappaB. Based on inhibitor studies, we hypothesize that the effect of A23187 on BLV expression appears to be mediated by PKC.     

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.     

No involvement of bovine leukemia virus in sporadic bovine lymphosarcoma

Using various portions of a molecularly cloned bovine leukemia virus (BLV) DNA as probes, the possible integration of a BLV genome or a BLV-related sequence into the chromosomal DNA of sporadic bovine leukosis (SBL) tumor cells was investigated by Southern blotting analysis. Under stringent as well as nonstringent conditions of hybridization, neither BLV nor BLV-related sequence specific to SBL DNAs was detected in any SBL tumor examined. These results provide conclusive evidence for lack of the relation of BLV or a BLV-related agent to SBL.     

Isolation of a monoclonal antibody which blocks vaccinia virus infection.

We have isolated a monoclonal antibody, B2, that neutralizes vaccinia virus infection. B2 reacts with a trypsin-sensitive cell surface epitope. B2 does not neutralize infection of herpes simplex virus, suggesting that the B2-reactive epitope is specifically involved in vaccinia virus entry. A survey of 12 different cell lines reveals a correlation between B2 reactivity and susceptibility to vaccinia virus infection. In addition, B2 interferes with vaccinia virus adsorption to target cells. Taken together, the B2-reactive epitope is part of a receptor that appears important for vaccinia virus entry.     

Integration of bovine leukemia virus DNA in the genomes of bovine lymphosarcoma cells

Integration of bovine leukemia virus (BLV) in the genomes of infected cells was investigated in cattle with enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). Southern blot hybridization of BLV cDNA to Eco RI and Xba I restriction fragments of EBL tumor DNAs revealed that: 1) one to four or more copies of proviral DNA were integrated per genome; 2) the restriction pattern of the integrated proviral DNA was the same in two or three different tumors from the same animals; and 3) different patterns were observed among tumors from four different animals. These findings suggest the monoclonal origin of different tumors in an individual animal and the existence of multiple chromosomal integration sites of BLV provirus. DNAs from several SBL tumors were also analyzed with the same restriction enzymes, but with both representative and cDNA3'-enriched's of BLV RNA. No hybridization bands reactive with representative BLV cDNA could be detected, while several bands appeared to hybridize with cDNA3'-enriched.