Bovine Leukemia Virus Envelope Research Articles

Long-term protection against bovine leukaemia virus replication in cattle and sheep.

In this report, we have evaluated the ability of two different types of live attenuated bovine leukaemia virus (BLV) variants (BLV DX and BLV 6073) to protect cattle and sheep against a heterologous wild-type BLV challenge. Four months after challenge, the protection of the vaccinated animals was effective in contrast to unvaccinated controls. However, long-term protection (18 months after challenge) was observed only in six out of seven animals, one of the vaccinated cattle being infected 12 months after challenge. A second prospective approach investigated the injection of naked plasmid DNA. Two sheep were injected with plasmid DNA encoding the BLV envelope proteins; the challenge virus infection was delayed but could not be completely abrogated. Our results demonstrate that vaccines based on live attenuated viruses and naked DNA injections are able to delay BLV infection, although complete protection cannot be achieved. In addition, our data cast light onto the need to perform long-term vaccination trials because challenge superinfection can occur even after apparent protection for 12 months.

Protection of cattle against bovine leukemia virus (BLV) infection could be attained by DNA vaccination.

The bovine leukemia virus (BLV) envelope gene encoding extracellular glycoprotein gp51 and transmembrane glycoprotein gp30 was cloned into a vehicle expression vector under the human cytomegalovirus (CMV) intermediate early promoter. The intramuscular injection of this plasmid vector generated a cellular immune response. Seven out of ten cows vaccinated with the DNA construct resisted a drastic challenge (500 BLV-infected lymphocytes as an infectious dose).

Up-regulation of tumor necrosis factor α mRNA is associated with bovine-leukemia virus (BLV) elimination in the early phase of infection

Protective immune responses were analyzed in eight sheep vaccinated with BLV envelope peptides and experimentally infected with bovine-leukemia virus (BLV). Five of eight peptide-immunized sheep showed a high T-cell proliferative response to the BLV peptides and all of these were protected from the infection. The other three peptide-immunized sheep showed no T-cell proliferative responses to any BLV antigens similar to control sheep, though they also exhibited resistance to BLV challenge. To investigate other mechanisms which suppress BLV expansion in these non-responding sheep, we measured the levels of the cytokine expressions before, and after, BLV challenge using competitive reverse-transcriptase polymerase chain-reaction systems. It was revealed that the expression of tumor necrosis factor α (TNFα) was higher in BLV-resistant sheep than in BLV-susceptible sheep. Thus, TNFα expression rather than specific T-cell activity may play an important role in the protective mechanism against BLV infection, at least during the primary viremia phase.

Bovine leukaemia virus envelope peptides cause immunomodulation in BALB/c mice

Immunomodulatory activity of two bovine leukaemia virus envelope (BLVEnv) derived peptides were examined in BALB/c mice. One is a peptide homologous to CKS-17 which is known as a 17-amino acid peptide derived from p15E of feline leukaemia virus (CKS-17/BLV), and the other is an 18-amino acid synthetic peptide of BLV Env 61-78 (pep61). Priming with CKS-17/BLV in vitro, as well as CKS-17, significantly suppressed the mitogen-induced proliferative responses of spleen cells in naive BALB/c mice. In addition, priming of spleen cells with pep61 in vitro and in vivo resulted in suppression of lipopolysaccaride-induced B-cell proliferative response. This suppression was partially due to the basic amino acid sequence in the peptide because if the pep61-derived peptide lacking Arg was used, this inhibitory activity was partially restored. In contrast, pep61 enhanced both concanavalin A-stimulated proliferative response and IL-2 production. These findings showed that pep61 may contribute to the modification of the host immune responses in the course of BLV infection.

Characterization of immune responses caused by bovine leukemia virus envelope peptides in sheep.

To study the immunomodulative activity caused by bovine leukemia virus envelope (BLV Env) peptide, sheep were immunized with two kinds of Th-epitope peptides, peptide 98 (BLV Env 98-117), and 61 (BLV Env 61-78). Four of eight immunized sheep showed specific proliferative responses against both of the peptide stimulations. To characterize the cells responding to the peptides, peptide-specific cells were established from the responding sheep by the continuous stimulation of peripheral blood mononuclear cells (PBMCs) with either peptide 98 or 61 in vitro. The peptide 98-specific cells consisted of CD4-positive cells, whereas the peptide 61-specific cells consisted of CD8-positive cells and MHC class II-positive cells. In addition, cytokine profile analysis indicated that the peptide 98-stimulated cells expressed IFN-gamma but not IL-10, although the peptide 61-stimulated cells expressed IL-10 but not IFN-gamma. These results show that BLV envelope peptides 98 and 61 can modulate immune responses of sheep lymphocytes in different ways and may contribute to the pathogenesis of BLV infection.

Conservative Mutations in the Immunosuppressive Region of the Bovine Leukemia Virus Transmembrane Protein Affect Fusion but Not Infectivity in Vivo

Many retroviruses, including bovine leukemia virus (BLV), contain a highly conserved region located about 40 amino acids downstream from the fusion peptide within the sequence of the external domain of the transmembrane (TM) protein. This region is notably thought to be involved in the presentation of the NH2-terminal peptide to allow cell fusion. By using hydrophobic cluster analysis and by analogy with the influenza A hemagglutinin structures, the core of the TM structure including this particular region was predicted to consist, in the BLV and other retroviral envelope proteins, of an alpha-helix followed by a loop region, both docked against a subsequent alpha-helix that forms a triple-stranded coiled coil. The loop region could undergo, as in hemagglutinin, a major refolding into an alpha-helix integrating the coiled coil structure and putting the fusion peptide to one tip of the molecule. Based on this model, we have identified amino acids that may be essential to the BLV TM structure, and a series of mutations were introduced in the BLV env gene of an infectious molecular clone. A first series of mutations was designed to disturb the coiled coil structure (substitutions with proline residues), whereas others would maintain the general TM structure. When expressed by Semliki Forest virus recombinants, all the mutated envelope proteins were stable and efficiently synthesized in baby hamster kidney cells. Both proline-substituted and conservative mutants were strongly affected in their capacity to fuse to CC81 indicator cells. In addition, it appeared that the integrity of the TM coiled coil structure is essential for envelope protein multimerization, as analyzed by metrizamide gradient centrifugation. Finally, to gain insight into the role of this coiled coil in the infectious potential of BLV in vivo, the mutated TM genes were introduced in an infectious and pathogenic molecular clone and injected into sheep. It appeared that only the conservative mutations (A60V and A64S) allowed maintenance of viral infectivity in vivo. Since these mutations destroyed the ability to induce syncytia, we conclude that efficient fusion capacity of the recombinant envelopes is not a prerequisite for the infectious potential of BLV in vivo. Viral propagation of these mutants was strongly affected in some of the infected sheep. However, the proviral loads within half of the infected animals (2 out of 2 for A60V and 1 out of 4 for A64S) were close to the wild-type levels. In these sheep, it thus appears that the A60V and A64S mutants propagate efficiently despite being unable to induce syncytia in cell culture.

Comparative processing of bovine leukemia virus envelope glycoprotein gp72 by subtilisin/kexin-like mammalian convertases

Intracellular proteolytic processing of bovine leukemia virus (BLV) envelope glycoprotein precursor (gp72) at the C-terminal end of the RVRR 268↓ site is an essential step for virus infectivity. Subtilisin/kexin-like convertases cleave proproteins at preferred RX(K/R)R↓ sites, including those commonly found in viral envelope glycoprotein precursors. We first demonstrated that gp72 is processed into gp51/gp30 in both CV1 cells and the furin-deficient LoVo cells, leading us to compare the ability of mammalian convertases to cleave BLV gp72 in vitro. In contrast to the inability of the neuroendocrine PC1 to cleave gp72, the convertases furin, PACE4, PC5-A and PC5-B, which process constitutively secreted precursors, can effectively cleave gp72 into gp51/gp30. N-terminal sequence analysis of the convertase-generated gp30 demonstrated that cleavage occurs at the in vivo-utilized RVRR↓SPV site. Such furin-, PACE4- and PC5-mediated processing was completely inhibited by the α 1-antitrypsin variant α1-PDX. Mutagenesis of the gp72 cleavage site into RVR G - T PV resulted in complete abrogation of gp72 processing by endogenous CV-1 cells and by convertases in vitro. Since our in vitro data suggest a redundancy in the ability of the convertases to cleave gp72, RT-PCR analysis was used to define the convertases expressed in B-lymphocytes, representing one of the major targets of BLV infection. Our data revealed that only furin and the newly discovered PC7 mRNAs are expressed in Raji, B-Jab and LG2 cell lines.

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-γ 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.

Identification of functional sites on bovine leukemia virus envelope glycoproteins using structural and immunological data

Sequence analysis using the sensitive hydrophobic cluster analysis method shows that the bovine leukemia virus envelope glycoproteins conserve the general organization of the influenza hemagglutinin into a 'stem', containing the external part of the transmembrane glycoprotein and the N-terminal and C-terminal parts of the external glycoprotein, and a 'head', containing only external glycoprotein residues. However, our analysis suggests, for the first time, that the bovine leukemia virus envelope head will not adopt the typical 'jelly-roll' fold of the influenza A hemagglutinin head, but most likely folds into another type of 'Greek-key' structure corresponding to the overall topology of constant immunoglobulin domains. We constructed a three-dimensional model for the bovine leukemia virus envelope head by homology modeling using the crystal structure of the human histocompatibility antigen HLA-A2 alpha 3 domain. Furthermore, we propose a general model for the oligomeric organization of this head, based on the hemagglutinin trimer. The proposed structural organization of bovine leukemia virus external glycoprotein is further supported by antipeptide and monoclonal antibody reactivities. Our modeling study suggests that the loops of the two neutralizing peptides located in the head are adjacent at the top of the domain and define a potential interaction site of the external glycoprotein with its cellular receptor. This site is topologically similar to the binding site of hemagglutinin with its cellular receptor, sialic acid. The other neutralizing peptides are located within a small domain linking the head to the stem. These data are of interest for defining other oncoviral glycoproteins heads and receptor-binding sites.

The (YXXL/I)2 signalling motif found in the cytoplasmic segments of the bovine leukaemia virus envelope protein and Epstein-Barr virus latent membrane protein 2A can elicit early and late lymphocyte activation events.

The cytoplasmic domains of the transducing subunits associated with B and T cell antigen receptors contain a common amino acid motif consisting of two precisely spaced Tyr-X-X-Leu/Ile sequences (where X corresponds to a variable residue). Expression of a single copy of this motif suffices to initiate B or T cell activation. The bovine leukaemia virus (BLV) is a B cell lymphotropic retrovirus which causes a non-neoplasic proliferation of B cells. The cytoplasmic domain of the BLV transmembrane envelope glycoprotein, gp30, possesses two overlapping copies of the Tyr-X-X-Leu/Ile-containing motif which could participate in the induction of B cell activation. Similarly, the N-terminal cytoplasmic domain of the latent membrane protein 2A (LMP2A) of the Epstein-Barr virus (EBV) contains a single copy of the Tyr-X-X-Leu/Ile-containing motif which could play a critical role in B cell transformation. To determine whether these two virus-encoded cytoplasmic domains are endowed with signalling functions, we constructed chimeric proteins by replacing the cytoplasmic tail of CD8-alpha with that of either BLV gp30 or EBV LMP2A. We show here that, once separately expressed in B or T cell lines, these chimeras are capable of triggering both calcium responses and cytokine production when cross-linked with an antibody to CD8-alpha. Furthermore, using site-directed mutagenesis, we demonstrated unequivocally that this signalling function may be accounted for by the Tyr-X-X-Leu/Ile motifs they contain.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of bovine leukemia virus proviral DNA in individual cells.

We have developed a method of analyzing individual cells to detect proviral DNA of the bovine leukemia virus (BLV) using flow cytometry and PCR. Individual cells of the BL3* cell line, which contain multiple integrated copies of the BLV provirus, and the uninfected cell line BL3(0), were sorted into wells of a 96-well plate. Following cell lysis, portions of the BLV envelope (ENV) and cellular prolactin (PRL) genes were amplified simultaneously using PCR. Viral and cellular products of first-round PCR were amplified separately in a second round of PCR using "heminested" primers. Separation of the PCR products by polyacrylamide gel electrophoresis yielded distinct fragments of the predicted sizes. The operational sensitivity of this method for the detection of virus was > 90% when testing single infected cells. In addition, we were able to reliably amplify DNA from a single BL3* cell among as many as 10(5) BL3(0) cells and established that the sensitivity for detecting a single infected cell among 20, 100, or 1000 uninfected cells was at least 90%. Estimates of low percentages of infected cells were obtained by applying probability theory to results of experiments conducted on wells containing more than one cell. Using these methods, B lymphocytes obtained from the peripheral blood of BLV-infected cattle were tested for proviral DNA. BLV ENV was identified in 76.9 +/- 4.9% of single B cells tested from a seropositive animal with persistent lymphocytosis (PL), but in only 0.033 +/- 0.009% of B cells from another seropositive cow without PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of sheep against bovine leukemia virus (BLV) infection by vaccination with recombinant vaccinia viruses expressing BLV envelope glycoproteins: correlation of protection with CD4 T-cell response to gp51 peptide 51-70.

We have previously constructed vaccinia virus (VV) recombinants containing a complete or truncated envelope (env) gene of bovine leukemia virus (BLV). Only recombinants carrying the complete env gene (VV-BLV2 and VV-BLV3) expressed env glycoprotein on the surface of virus-infected cells and produced an antibody response in rabbits. In the present study, these VV recombinants were used to immunize sheep prior to challenge with BLV-infected peripheral blood mononuclear cells. Both humoral and cell-mediated immunity were monitored in infected animals. Sheep inoculated with recombinants containing the complete env gene showed a CD4 response to a defined epitope of gp51, but this response was absent 4 months postchallenge. Anti-gp51 antibodies appeared in animals inoculated with complete env 2 weeks after challenge, reached a peak at 4 weeks, and subsequently declined over 16 months. No CD4 response was recorded in animals inoculated with recombinants containing truncated env gene (VV-BLV1). BLV-infected control animals and those animals receiving VV-BLV1 were slower to develop antibodies postchallenge, and the titers of anti-gp51 antibodies continued to increase over 16 months. Proviral DNA was detected by the polymerase chain reaction in the four groups at 6 weeks after challenge. However, it could not be detected 4 months postinfection in the VV groups inoculated with complete env. Provirus was present in the VV-BLV1 and control groups over the 16-month trial period. These results demonstrate that vaccination with VV recombinants containing the complete env gene of BLV protects sheep against infection and that protection correlated with a CD4 T-cell response to a defined epitope.

Involvement of CD8+ T cells in delayed-type hypersensitivity responses against bovine leukemia virus (BLV) induced in sheep vaccinated with recombinant vaccinia virus expressing BLV envelope glycoprotein.

Delayed-type hypersensitivity responses against bovine leukemia virus (BLV) envelope glycoprotein (gp60) were induced in the skin of sheep vaccinated with recombinant vaccinia virus (RVV) expressing BLV glycoprotein. The lesions were characterized by marked infiltration of lymphocytes, slight migration of neutrophils, eosinophils, and macrophages in the dermis to hypodermis, and partial intercellular edema in the reticular layer. Immunohistochemical analysis with monoclonal antibodies demonstrated that the lymphocytic infiltrates consisted mainly of CD8+ T cells (53.7-55.8% at 48 hours post-challenge of BLV), CD4+ T cells (24.7-26.7%), and B cells (11.5-16.9%). The role of CD4+ and CD8+ T cells in suppressing BLV growth in RVV-vaccinated animals is discussed.

Enhancement of Bovine Leukemia Virus-Induced Syncytia Formation by Di- and Tripeptides

Short hydrophobic peptides were previously shown to inhibit infectivity of para- and orthomyxoviruses. We tested the ability of a series of di- and tripeptides to interfere with cell fusion induced by bovine leukemia virus (BLV). Peptides containing a hydrophobic contribution and/or a positive net charge strongly enhanced syncytia formation induced by BLV on CC81 indicator cells. The size of the multinucleated cells was strongly increased (up to 10-fold) in the presence of the enhancer peptides whereas no effect was observed on the indicator cells in the absence of BLV. The peptides thus amplified the fusion process initiated by BLV envelope glycoproteins. The effect was dose-dependent at concentrations ranging from 20 to 640 μM and did not result from an increased expression of BLV proteins. The peptides did not compete with anti-gp51 monoclonal antibodies for the recognition of eight well-defined epitopes of gp51. We consequently hypothesize that the enhancer peptides interact with the membrane of BLV-producing cells and/or indicator cells and propose a model based on molecular modeling.

Augmentation of bovine leukemia virus (BLV)-specific lymphocyte proliferation responses in ruminants by inoculation with BLV env-recombinant vaccinia virus: their role in the suppression of BLV replication.

Lymphocyte proliferation responses were investigated in sheep and cattle, in which the replication of bovine leukemia virus (BLV) had been known to be suppressed by inoculation with recombinant vaccinia virus (rVV) expressing BLV envelope glycoprotein (gp60). Enhanced lymphocyte proliferation responses were observed in animals inoculated with rVV, regardless of whether they were naive or BLV carriers. These responses were roughly inversely correlated to the growth of BLV in the peripheral blood leukocytes. In contrast, there was no apparent correlation between humoral immune response and BLV growth. Based on these results, it was suggested that rVV rendered its suppressive effect of BLV replication primarily via augmentation of cell-mediated immunity.

Fusogenic segments of bovine leukemia virus and simian immunodeficiency virus are interchangeable and mediate fusion by means of oblique insertion in the lipid bilayer of their target cells.

Modified bovine leukemia virus (BLV) glycoproteins were expressed by using vaccinia virus recombinants, and their fusogenic capacities were examined by a syncytia-formation assay. This analysis indicates that (i) both BLV envelope glycoproteins gp51 and gp30 are necessary for cell fusion; (ii) insertion of the N-terminal segment of gp30 (fusion peptide) into the lipid bilayer in an oblique orientation, as predicted by computer conformational analysis, results in fusogenic capacities higher than insertion in a perpendicular or parallel orientation; and (iii) replacement of the BLV fusion peptide with its simian immunodeficiency virus counterpart does not modify the fusogenic capacity of the BLV glycoprotein.

Iodoacetamide treatment of bovine leukemia virus glycoprotein gp51 enhances the Western blotting reactivity of anti-peptide antibodies

Rabbit polyclonal antibodies were raised against synthetic peptides of the bovine leukemia virus envelope glycoprotein gp51 and tested against the full size protein by the Western blotting technique. We show that acetylation of gp51 by iodoacetamide either maintains or significantly increases the antigen-antibody reaction and conclude therefrom that the reactive potential of an anti-peptide antibody may require acetylation of the sulfhydryl groups of the blotted protein.

Use of synthetic peptides to map sequential epitopes recognized by monoclonal antibodies on the bovine leukemia virus external glycoprotein

Six sequential epitopes (A, B, B′, D, D′, E) were previously defined on the bovine leukemia virus (BLV) envelope glycoprotein gp51 by their reactivity with monoclonal antibodies. A panel of synthetic peptides covering almost the entire sequence of gp51 was tested in enzyme-linked immunosorbent assays in order to localize these epitopes. E was shown to be included in peptide 142–161 (MCF4), B and B′ in peptide 195–205, D and D′ in peptide 218–237 (MCF6), and A in peptide 249–268 (MCF7). These results extend and confirm previous observations suggesting that the sequential epitopes recognized by our battery of monoclonal antibodies are located in the carboxylic half of BLV gp51.

Protective immunity against bovine leukaemia virus (BLV) induced in carrier sheep by inoculation with a vaccinia virus-BLV env recombinant: association with cell-mediated immunity.

The effects of vaccination of sheep with a recombinant vaccinia virus (rVV) expressing the bovine leukaemia virus (BLV) envelope glycoprotein (gp60) were studied by determining BLV titres in peripheral blood leukocytes after vaccination and challenge. The proliferation of BLV was suppressed markedly, not only when rVV was inoculated prior to challenge with BLV, but also when it was inoculated after challenge. These results indicate that vaccination with rVV induces protective immunity that can suppress the growth of BLV in carrier animals. Since rVV induced a strong anti-BLV delayed-type hypersensitivity response without producing detectable levels of binding or neutralizing antibodies, and there was no apparent correlation between the humoral immune response and BLV proliferation, a cell-mediated immune response was assumed to play a major role in protective immunity.

Recombinant vaccinia virus expression of the bovine leukaemia virus envelope gene and protection of immunized sheep against infection

The bovine leukaemia virus (BLV) envelope gene encoding extracellular glycoprotein gp51 and transmembrane glycoprotein gp30 was cloned into the HA locus of vaccinia virus (Copenhagen strain), downstream of the vaccinia virus early-late promoter, H6, or a triple promoter element consisting of the promoter for the vaccinia virus H6 gene, the promoter for the cowpox virus A-type inclusion (AT1) gene and the promoter for the vaccinia virus HA gene. Inoculation of rabbits or sheep with the recombinant vaccinia virus coding for gp51 and gp30 or an uncleaved env precursor induced neutralizing antibodies to BLV. These antibodies competed with monoclonal antibodies directed against gp51 epitopes F, G, and H previously shown to be of crucial importance for BLV infection. Seven out of eight sheep vaccinated with the vaccinia recombinants resisted a drastic challenge (1.5 × 10 3 sheep infectious doses) with BLV-infected lymphocytes. These results show that vaccination with BLV env vaccinia recombinants protects sheep against infection with extremely high doses of BLV-infected heterologous lymphocytes.