A. Burny

Tumor-selective gene transduction and cell killing with an oncotropic autonomous parvovirus-based vector.

A recombinant MVMp of the fibrotropic strain of minute virus of mice (MVMp) expressing the chloramphenicol acetyltransferase reporter gene was used to infect a series of biologically relevant cultured cells, normal or tumor-derived, including normal melanocytes versus melanoma cells, normal mammary epithelial cells versus breast adenocarcinoma cells, and normal neurons or astrocytes versus glioma cells. As a reference cell system we used normal human fibroblasts versus the SV40-transformed fibroblast cell line NB324K. After infection, we observed good expression of the reporter gene in the different tumor cell types, but only poor expression if any in the corresponding normal cells. We also constructed a recombinant MVMp expressing the green fluorescent protein reporter gene and assessed by flow cytometry the efficiency of gene transduction into the different target cells. At a multiplicity of infection of 30, we observed substantial transduction of the gene into most of the tumor cell types tested, but only marginal transduction into normal cells under the same experimental conditions. Finally, we demonstrated that a recombinant MVMp expressing the herpes simplex virus thymidine kinase gene can, in vitro, cause efficient killing of most tumor cell types in the presence of ganciclovir, whilst affecting normal proliferating cells only marginally if at all. However, in the same experimental condition, breast tumor cells appeared to be resistant to GCV-mediated cytotoxicity, possibly because these cells are not susceptible to the bystander effect. Our data suggest that MVMp-based vectors could prove useful as selective vehicles for anticancer gene therapy, particularly for in vivo delivery of cytotoxic effector genes into tumor cells.

The major homology region of bovine leukaemia virus p24gag is required for virus infectivity in vivo.

In order to gain insight into the role of the major homology region (MHR) in the infectious potential of bovine leukaemia virus (BLV), mutations were introduced into the capsid gene of an infectious molecular clone. A provirus that was designed to contain only a slightly modified version of the MHR (substitution of phenylalanine 147 with a tyrosine) was still infectious in vivo. Furthermore, the provirus loads were not significantly different from those obtained with a wild-type virus. A second mutant was designed to analyse a mild modification of the MHR at the level of arginine 150. The substitution of this residue with a lysine completely destroyed the infectious potential of the recombinant virus. Finally, a third mutant that was deleted in the MHR region was unable to infect the host. Thus it appears that the integrity of the MHR domain is essential for BLV infectivity in vivo.

FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/macrophages.

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers.

Striking similarities between HIV-1 Env protein and the apoptosis mediating cell surface antigen Fas. Role in the pathogenesis of AIDS

We have designed a computer strategy in order to detect systematically peptidic sites with the potential of interfering with the immune regulatory processes. Applying this software to HIV-1 proteins has led us to unravel a few peptidic sites which could either act directly or be the targets of an auto-immune reaction during HIV-1 infection. We previously reported that the SLWDQ pentapeptide identity with a critical site of CD4 could trigger in HIV-1 infected individuals both an humoral and a cellular autoimmune reaction. In this study, we focused on surprising similitudes unravelled by our software Automat, between HIV-1/2 and another immunoregulatory molecule, the Fas protein which is also called the apoptosis-mediating cell-surface antigen.

Isolation of the missing 5'-end of the encoding region of the bovine leukemia virus cell receptor gene.

SummaryThe missing 5′-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5′) was isolated from a lambda gt11 cDNA library using the32P-labeledEcoRI-SamI fragment corresponding to the 5′-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5′ cDNA revealed that the 1058 bpEcoRI fragment at its 5′-end contained a new 114 amino acid long sequence, and at its 3′-end contained a completely identical 88 amino acid overlapping region with the 5′-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venters tag sequences of an unidentified gene from the human brain library.

Leukemogenesis by bovine leukemia virus (BLV)

Bovine leukemia virus (BLV) is a type C retrovirus which is the causative agent of a B cell lymphocytic leukemia horizontally transmitted in cattle (1, 2, 3). This virus was proven to be totally exogenous to its target animal as well as to sheep, goat, mouse, cat, chicken and man (4). It appears to be unrelated, as judged by nucleic acid hybridization and immunological properties, to any other retrovirus tested up to now. However, a slight relatedness between the amino terminal sequence of the major internal protein (p24) of BLV and the corresponding polypeptide (p30) of a murine type C virus has recently been uncovered (5). This suggests the existence of a remote common ancestor for both viruses.

The bovine leukemia virus tax gene contains an enhancer sequence

Transactivator proteins of the bovine leukemia (BLV) and human T-lymphotropic (HTLV) viruses increase long terminal repeat (LTR)-directed viral gene expression and act as immortalizing oncogenes in tissue culture. We report here that the BLV tax gene sequence contains an enhancer-like activity. The X long open reading frame was cloned up-stream of the beta-globin promoter linked to the chloramphenicol acetyltransferase (CAT) gene. In the presence of tax sequences, up to sevenfold enhancement of CAT expression was observed. A computer-assisted homology search revealed the presence of a consensus enhancer core sequence (GTGTTGTTGGTTG) into the third exon of the 2.1-kb X mRNA. These studies demonstrate that the tax gene contains a transcriptional enhancer which could be involved in early viral gene expression in vivo.

Absence probable de la translocation robertsonienne 1/29 en race bovine Blanc Bleu Belge

La translocation robertsonienne 1/29, anomalie chromosomique de structure, intervient frequemment comme cause dinfertilite. Nous avons donc voulu connaitre sa frequence en race bovine Blanc Bleu Belge, jusqualors non etudiee pour ce facteur. Au sein dun echantillon de 138 taureaux de race Blanc Bleu Belge, la translocation 1/29 na pas pu etre identifiee. Nous pouvons estimer avec 5% de risque derreur que la frequence de cette translocation au sein de la population Blanc Bleu Belge doit etre inferieure a 0,69%. Ce resultat trouve une explication dans lorigine de cette race. En effet, le Blanc Bleu Belge est issu dun melange de races laitieres locales et de la race Shorthorn, lesquelles peuvent etre a priori considerees comme indemnes de la translocation 1/29.

Identification of the bovine leukemia virus transactivating protein (p34x).

Enzootic bovine leukosis (EBL) has been recognized as a neoplasm of infectious origin for half a century. The agent, bovine leukemia virus (BLV), is a retrovirus discovered in 1969 in short-term cultures of peripheral lymphocytes from animals with persistent lymphocytosis, a benign response to BLV infection. A virus distantly related to BLV was more recently identified as the etiological agent in the vast majority of cases of adult T cell leukemia and named for that reason human T-lymphotropic virus I (HTLV-I) [20]. The pathologies of BLV- and HTLV-I-in-duced diseases are notably similar, namely absence of chronic viremia, a long latency period, and lack of preferred integration sites in tumors. A second human virus, called HTLV-II, was identified in the Mo T cell line, derived in 1976 from the spleen of a patient with T cell-variant hairy cell leukemia [2, 10]. Other isolates of HTLV-I and -II have since been obtained around the world. Both viruses not only transform normal T-lymphocytes but might also very well be involved in a number of degenerative diseases of the nervous system.

Bovine leukemia virus, a distinguished member of the human T-lymphotropic virus family

Bovine leukemia virus (BLV) has many structural and functional characteristics in common with the human T-lymphotropic viruses (HTLVs). The most striking feature of these retroviruses is the existence of a long open reading frame (LOR) located at the 3 side of the provirus between the right end of the 3 side of env gene and the left end of the long terminal repeat (LTR). It is believed that the LOR protein product is of critical importance in the induction process of the tumor phase of bovine leukemia. Prevention of BLV infection will be attempted by vaccination. To that aim, careful study of BLV envelope glycoprotein epitopes has shown that epitopes F, G, and H play a major role in biological activities of the virus. Their native structure depends upon glycosylation of the peptide backbone.