Inhibition of bovine sperm-zona binding by bovine herpesvirus-1.

The purpose of the present study was to identify a potential interference of bovine herpesvirus-1 (BoHV-1) with sperm-oocyte interactions during bovine in vitro fertilization. An inhibition of almost 70% of sperm-zona binding was observed when bovine cumulus-denuded oocytes were inseminated in the presence of 10(7) 50% tissue culture infective dose/ml BoHV-1. The inhibitory effect of BoHV-1 on sperm-zona binding was mediated by an interaction of the virus with spermatozoa, but not with oocytes. Treatment of spermatozoa with BoHV-1, however, did not affect sperm motility and acrosomal status. Antiserum against BoHV-1 prevented the virus-induced inhibition of sperm-zona binding, indicating that BoHV-1 itself affects the fertilization process. In order to investigate which BoHV-1 glycoprotein(s) are responsible for the virus-sperm interaction, BoHV-1 was treated with monoclonal antibodies against the viral glycoproteins gB, gC, gD and gH prior to insemination. Anti-gC completely prevented the inhibitory effect of BoHV-1 on sperm-zona binding, while anti-gD caused a reduction of this inhibition. Further evidence for the involvement of gC and gD in the virus-sperm interaction was provided by the fact that purified gC and gD decreased sperm-zona binding in a dose-dependent way with gC being more effective than gD. These results indicated that BoHV-1 inhibits bovine sperm-zona binding by interacting with spermatozoa. The binding of BoHV-1 to a spermatozoon is mediated by the viral glycoproteins gC and gD, and therefore seems to be comparable with the mechanisms of BoHV-1 attachment to its natural host cell.

Effects of hypervaccination with bovine herpesvirus type 1 gE-deleted marker vaccines on the serological response and virological status of calves challenged with wild-type virus.

Twenty-four calves were immunised four times with gE-deleted infectious bovine rhinotracheitis marker vaccines before being challenged with small doses of wild-type bovine herpesvirus type 1 (BHV-1). The repeated vaccinations induced strong immunity that prevented detectable virus replication and gE-seroconversion after the challenge infection in most of the calves. The hypervaccinated calves that shed virus after the challenge infection showed no delay in gE-seroconversion compared with unvaccinated control calves. Using a sensitive nested PCR, BHV-1 gE sequences could be detected in the trigeminal ganglia of several of the gE-seronegative, challenge-infected calves, possibly indicating the presence of wild-type BHV-1 DNA.

Isolation of a glycoprotein E-deleted bovine herpesvirus type 1 strain in the field.

During a field trial to evaluate the efficacy of repeated vaccinations with bovine herpesvirus type 1 (BHV-1) marker vaccines, a glycoprotein E (gE)-negative BHV-1 strain was isolated from the nasal secretions of two cows, eight months after vaccination with a gE-negative live-attenuated vaccine, initially given intranasally, then intramuscularly. The strain isolated was characterised using immunofluorescence, restriction analysis and PCR. All the techniques used identified the isolated virus as a gE-negative BHV-1 phenotypically and genotypically identical to the Za strain used as a control.

Inhibition of histone deacetylases induces bovine leukemia virus expression in vitro and in vivo.

Packaging into nucleosomes results in a global transcriptional repression as a consequence of exclusion of sequence-specific factors. This inhibition can be relieved by using inhibitors of histone deacetylases, acetylation being a major characteristic of transcriptionally active chromatin. Paradoxically, the expression of only approximately 2% of the total cellular genes is modulated by histone hyperacetylation. To unravel the potential role of this transcriptional control on BLV expression, we tested the effect of two highly specific inhibitors of deacetylases, trichostatin A (TSA) and trapoxin (TPX). Our results demonstrate that treatment with TSA efficiently enhanced long terminal repeat-directed gene expression of integrated reporter constructs in heterologous D17 stable cell lines. To further examine the biological relevance of these observations made in vitro, we analyzed ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected sheep. TSA deacetylase inhibitor induced a drastic increase in viral expression at levels comparable to those induced by treatment with phorbol-12-myristate 13-acetate and ionomycin, the most efficient activators of BLV expression known to date. TSA acted directly on BLV-infected B lymphocytes to increase viral expression and does not seem to require T-cell cooperation. Inhibition of deacetylation after treatment with TSA or TPX also significantly increased viral expression in PBMCs from cattle, the natural host for BLV. Together, our results show that BLV gene expression is, like that of a very small fraction of cellular genes, also regulated by deacetylation.

Genetic and antigenic variability in bovine viral diarrhea virus (BVDV) isolates from Belgium.

This report describes the genetic and antigenic variability of bovine viral diarrhea virus strains isolated in Belgium. Part of the 5' untranslated region and the 5' end of the gp53 (E2) coding sequence were amplified by PCR and sequenced. Phylogenetic analysis showed that most field isolates segregated into genotypes Ib or II. Only one out of 28 field isolates belonged to genotype Ia. Interestingly, some type I strains were equally divergent from types Ia and Ib strains and clustered into additional subtypes within genotype I. Immune sera from young calves experimentally inoculated with field isolates first identified on the basis of their sequences were used in two-way neutralisation experiments. The results clearly differentiated type I from type II strains although some degree of cross-neutralisation was observed. Within type I, the new clusters could not be antigenically differentiated from the more prevalent type Ib strains or from type Ia strain NADL, suggesting that BVDV genotype I is antigenically homogeneous. The isolation of BVDV types I and II strains from cell lines and from a bovine vaccine suggest that molecular epidemiology surveillance is warranted for BVDV.

Characterization of monoclonal antibodies directed against the bovine herpesvirus-1 glycoprotein E and use for the differentiation between vaccinated and infected animals.

A panel of seven monoclonal antibodies (MAbs) directed against the bovine herpesvirus-1 (BHV-1) glycoprotein E (gE) was obtained. For that purpose, mice were either tolerized to BHV-1 gE-negative virus and then immunized with wild type BHV-1 or immunized with plasmid DNA expressing the gE and gI glycoproteins. The MAbs were characterized by their reactivity with the gE protein or the gE/gI complex and by competition experiments. Results showed that the MAbs were directed against three antigenic domains, two located on the gE glycoprotein and one on the gE/gI complex. Blocking experiments were performed with sera from experimentally vaccinated and infected cattle. A competition was observed between gE-positive bovine sera and six of the seven MAbs. The bovine sera thus recognized two of the three antigenic sites. Field sera were then tested in blocking enzyme-linked immunosorbent assay using one horseradish peroxidase-conjugated MAb. A specificity of 98.2% and a sensitivity of 98.2% compared to the commercially available test were observed.

Suboptimal enhancer sequences are required for efficient bovine leukemia virus propagation in vivo: implications for viral latency.

Repression of viral expression is a major strategy developed by retroviruses to escape from the host immune response. The absence of viral proteins (or derived peptides) at the surface of an infected cell does not permit the establishment of an efficient immune attack. Such a strategy appears to have been adopted by animal oncoviruses such as bovine leukemia virus (BLV) and human T-cell leukemia virus (HTLV). In BLV-infected animals, only a small fraction of the infected lymphocytes (between 1 in 5,000 and 1 in 50,000) express large amounts of viral proteins; the vast majority of the proviruses are repressed at the transcriptional level. Induction of BLV transcription involves the interaction of the virus-encoded Tax protein with the CREB/ATF factors; the resulting complex is able to interact with three 21-bp Tax-responsive elements (TxRE) located in the 5' long terminal repeat (5' LTR). These TxRE contain cyclic AMP-responsive elements (CRE), but, remarkably, the "TGACGTCA" consensus is never strictly conserved in any viral strain (e.g.,AGACGTCA, TGACGGCA, TGACCTCA). To assess the role of these suboptimal CREs, we introduced a perfect consensus sequence within the TxRE and showed by gel retardation assays that the binding efficiency of the CREB/ATF proteins was increased. However, trans-activation of a luciferase-based reporter by Tax was not affected in transient transfection assays. Still, in the absence of Tax, the basal promoter activity of the mutated LTR was increased as much as 20-fold. In contrast, mutation of other regulatory elements within the LTR (the E box, NF-kappa B, and glucocorticoid- or interferon-responsive sites [GRE or IRF]) did not induce a similar alteration of the basal transcription levels. To evaluate the biological relevance of these observations made in vitro, the mutations were introduced into an infectious BLV molecular clone. After injection into sheep, it appeared that all the recombinants were infectious in vivo and did not revert into a wild-type virus. All of them, except one, propagated at wild-type levels, indicating that viral spread was not affected by the mutation. The sole exception was the CRE mutant; proviral loads were drastically reduced in sheep infected with this type of virus. We conclude that a series of sites (NF-kappa B, IRF, GRE, and the E box) are not required for efficient viral spread in the sheep model, although mutation of some of these motifs might induce a minor phenotype during transient transfection assays in vitro. Remarkably, a provirus (pBLV-Delta 21-bp) harboring only two TxRE was infectious and propagated at wild-type levels. And, most importantly, reconstitution of a consensus CRE, within the 21-bp enhancers increases binding of CREB/ATF proteins but abrogates basal repression of LTR-directed transcription in vitro. Suboptimal CREs are, however, essential for efficient viral spread within infected sheep, although these sites are dispensable for infectivity. These results suggest an evolutionary selection of suboptimal CREs that repress viral expression with escape from the host immune response. These observations, which were obtained in an animal model for HTLV-1, are of interest for oncovirus-induced pathogenesis in humans.

CD154 costimulated ovine primary B cells, a cell culture system that supports productive infection by bovine leukemia virus.

Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and gamma-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.

Genetic determinants of bovine leukemia virus pathogenesis.

The understanding of HTLV-induced disease is hampered by the lack of a suitable animal model allowing the study of both viral replication and leukemogenesis in vivo. Although valuable information has been obtained in different species, such as rabbits, mice, rats, and monkeys, none of these systems was able to conciliate topics as different as viral infectivity, propagation within the host, and generation of leukemic cells. An alternate strategy is based on the understanding of diseases induced by viruses closely related to HTLV-1, like bovine leukemia virus (BLV). Both viruses indeed belong to the same subfamily of retroviruses, harbor a similar genomic organization, and infect and transform cells of the hematopoietic system. The main advantage of the BLV system is that it allows direct experimentation in two different species, cattle and sheep.

Discordance between bovine leukemia virus tax immortalization in vitro and oncogenicity in vivo.

Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

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.

In vivo rescue of a silent tax-deficient bovine leukemia virus from a tumor-derived ovine B-cell line by recombination with a retrovirally transduced wild-type tax gene.

The lack of bovine leukemia virus (BLV) expression is a consistent finding in freshly isolated ovine tumor cells and in the B-cell lines derived from these tumors. In order to gain further insight into the mechanisms of BLV silencing in these tumors, we have used the YR2 B-cell line, which was derived from the leukemic cells of a BLV-infected sheep. This cell line contains a single, monoclonally integrated, silent provirus, which cannot be reactivated either by stimulation in vitro or by in vivo injection of the tumor cells or cloned proviral DNA in sheep. Sequence analysis of the tax gene from the YR2 cell line identified two G-to-A transitions (G7924 to A7924 and G8149 to A8149) that result in E-to-K amino acid changes at positions 228 and 303 in the Tax protein. Following retroviral vector-mediated transfer of a wild-type tax gene into YR2 cells, we showed that BLV mRNA, viral proteins, and virions were produced, demonstrating that the cellular factors required for virus expression were present in the original YR2 cell line. Injection of this transduced YR2 cell line in sheep led to the rescue of replication-competent BLV proviruses. The integrated competent proviruses exhibited unique chimeric tax genes, which arose from homologous recombination between the transduced wild-type tax and the YR2-derived tax sequences. Furthermore, in one of these functional recombinant proviruses, only the A8149-to-G8149 reversion was present, providing clear evidence that the defect underlying the silent phenotype in YR2 cells results from a single C-terminal E303-to-K303 amino acid substitution in the BLV Tax protein. Our observations suggest that a single strategically located mutation in tax provides a mechanism for BLV inactivation in B-cell tumors.

Bovine leukemia virus-induced persistent lymphocytosis in cattle does not correlate with increased ex vivo survival of B lymphocytes.

Bovine leukemia virus (BLV) is an oncogenic retrovirus associated with B-cell lymphocytosis, leukemia, and lymphosarcoma in the ovine and bovine species. We have recently reported that in sheep, BLV protects the total population of peripheral blood mononuclear cells (PBMCs) from ex vivo spontaneous apoptosis. This global decrease in the apoptosis rates resulted from both direct and indirect mechanisms which allow extension of cell survival. Although sheep are not natural hosts for BLV, these animals are prone to develop virus-induced leukemia at very high frequencies. Most infected cattle, however, remain clinically healthy. This difference in the susceptibilities to development of leukemia in these two species might be related to alterations of the apoptotic processes. Therefore, we designed this study to unravel the mechanisms of programmed cell death in cattle. We have observed that PBMCs from persistently lymphocytotic BLV-infected cows were more susceptible to spontaneous ex vivo apoptosis than cells from uninfected or aleukemic animals. These higher apoptosis rates were the consequence of an increased proportion of B cells exhibiting lower survival abilities. About one-third of the BLV-expressing cells did not survive the ex vivo culture conditions, demonstrating that viral expression is not strictly associated with cell survival in cattle. Surprisingly, culture supernatants from persistently lymphocytotic cows exhibited efficient antiapoptotic properties on both uninfected bovine and uninfected ovine cells. It thus appears that indirect inhibition of cell death can occur even in the presence of high apoptosis rates. Together, these results demonstrate that the protection against spontaneous apoptosis associated with BLV is different in cattle and in sheep. The higher levels of ex vivo apoptosis occurring in cattle might indicate a decreased susceptibility to development of leukemia in vivo.

Does dietary iron intake influence the iron status in hospitalised elderly patients?

PURPOSE: To investigate whether there is a relationship between dietary iron intake and body iron store in hospitalised elderly. METHODS: During a three months period, 163 consecutive patients older than 70 years and irrespective of their medical diagnosis participated in this study. Eight patients were excluded because of a lack of co-operation or a too bad general condition. Total food intake data were collected at admission by using a 7 days food-intake and nutrient-supplement record. The average daily dietary intake of each of the nutrients (total energy intake and potential inhibiting or enhancing factors such as calcium, dietary fibres and vitamin C) were calculated. Serum levels of ferritin, iron, transferrin, haemoglobin and mean corpuscular volume were also measured in the 155 study patients. RESULTS: A depleted iron store, defined as a serum ferritin level < 50 microg/L, was found in 23 patients (15%) of whom 17 were anaemic. The mean daily intake of iron (9.5 mg for the total group, 10 mg and 9.2 mg for men and women, respectively), vitamin C, calcium an dietary fibre was not significantly different between patients with depleted and adequate iron stores. The serum iron, transferrin, MCV, haemoglobin and ferritin levels were comparable between patients with a low (< 9 mg, n = 64) and normal (> or = 9 mg, n = 91) iron intake. We found no correlation between the dietary iron intake and the serum ferritin level (r = 0.06, p = 0.45), even not after exclusion of the patients with a serum ferritin level > 300 microg/L (r = 0.005, p = 0.95). CONCLUSION: We found no relationship between the dietary iron intake and biochemical indicators of the iron status in an elderly population. If no gastrointestinal or another organ-related cause might be found, iron deficiency can hardly be attributed to an impaired iron intake without a nutritional investigation. Other parameters may also play an important role in its multifactorial pathogenesis.

Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.

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.

In vitro and in vivo oncogenic potential of bovine leukemia virus G4 protein.

In addition to the genes involved in the structure of the viral particle, the bovine leukemia virus (BLV) genome contains a region called X which contains at least four genes. Among them, the tax and rex genes, respectively, are involved in transcriptional and posttranscriptional regulation of viral transcription. Two other genes, R3 and G4, were identified after cloning of the corresponding mRNAs from BLV-infected lymphocytes. Although the function of the two latter genes is still unknown, they appear to have important roles, since deletion of them restricts viral propagation in vivo. In order to assess the oncogenic potential of the R3 and G4 proteins, we first analyzed their ability to immortalize and/or transform primary rat embryo fibroblasts (Refs). In this assay, the G4 but not the R3 protein cooperated with the Ha-ras oncogene to induce tumors in nude mice. It thus appears that G4 exhibited oncogenic potential in vitro. To extend these observations in vivo, the pathology induced by recombinant viruses with mutations in G4 and in R3 and G4 was next evaluated with the sheep animal model. Viral propagation, as measured by semiquantitative PCR, appeared to be reduced when the R3 and G4 genes were deleted. These observations confirm and extend our previous data underlining the biological function of these genes. In addition, we present the results of a clinical survey that involves 39 sheep infected with six different BLV recombinants. Over a period of 40 months, 83% of the sheep infected with a wild-type virus developed leukemias and/or lymphosarcomas. In contrast, none out of 13 sheep infected with viruses with mutations in G4 or in R3 and G4 developed disease. We conclude that in addition to its oncogenic potential in vitro, G4 is required for pathogenesis in vivo. These observations should help us gain insight into the process of leukemogenesis induced by the related human T-cell leukemia virus type 1.

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.

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis.

Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo. Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV.

Lack of mutation in the WAF1/CIP1 gene during bovine leukemia virus-induced leukemogenesis.

As a cyclin-dependent kinases (Cdks) inhibitor (CDI), the protein p21WAF1/CIP1 is able to interfere with cell cycle progression. Its expression is upregulated by wild-type p53, and the p21WAF1/CIP1 protein appears to be a potent effector of the p53-dependent cell cycle regulatory pathway. We have previously reported that p53 mutations frequently occur during bovine leukemia virus (BLV)-induced leukemogenesis in cattle but not in sheep. Therefore, we have investigated the involvement of p21WAF1/CIP1 mutations in the tumorigenic process associated with BLV. We first cloned the bovine and ovine WAF1 genes and determined the complete nucleotide sequences of their second coding exons. These sequences share respectively 79% and 80% homology with those of the human counterpart exon. In order to screen for mutations that could be associated with BLV-induced pathogenicity, we performed single strand conformation polymorphism (SSCP) assays on the WAF1 genes from BLV-induced tumors. No WAF1 mutations were detected in any of the ten BLV-induced bovine tumor samples. Among eleven sheep tumors and three ovine cell lines, only one sample revealed a single mutation in the WAF1 coding sequence, but this mutation was silent at the translational level. We concluded that mutations of the WAF1 gene are not involved in the development of the tumors during BLV-induced leukemogenesis.