BLV-infected Sheep Research Articles

Chemoresistance to Valproate Treatment of Bovine Leukemia Virus-Infected Sheep; Identification of Improved HDAC Inhibitors.

We previously proved that a histone deacetylase inhibitor (valproate, VPA) decreases the number of leukemic cells in bovine leukemia virus (BLV)-infected sheep. Here, we characterize the mechanisms initiated upon interruption of treatment. We observed that VPA treatment is followed by a decrease of the B cell counts and proviral loads (copies per blood volume). However, all sheep eventually relapsed after different periods of time and became refractory to further VPA treatment. Sheep remained persistently infected with BLV. B lymphocytes isolated throughout treatment and relapse were responsive to VPA-induced apoptosis in cell culture. B cell proliferation is only marginally affected by VPA ex vivo. Interestingly, in four out of five sheep, ex vivo viral expression was nearly undetectable at the time of relapse. In two sheep, a new tumoral clone arose, most likely revealing a selection process exerted by VPA in vivo. We conclude that the interruption of VPA treatment leads to the resurgence of the leukemia in BLV-infected sheep and hypothesize that resistance to further treatment might be due to the failure of viral expression induction. The development of more potent HDAC inhibitors and/or the combination with other compounds can overcome chemoresistance. These observations in the BLV model may be important for therapies against the related Human T-lymphotropic virus type 1.

Viral expression directs the fate of B cells in BLV-infected sheep

There is a long lasting debate about the latency of human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). Evidence indicates that these viruses are transcriptionally silent and replicate through mitotic division of infected cells (clonal expansion). However, this model is inconsistent with the permanent and vigorous stimulation of the host immune response directed against these viruses. To address this apparent paradox, we studied the fate of cells in which viral expression was transiently induced. Using a dual fluorochrome labeling approach, we show that virus-positive and negative cell populations have different kinetics in BLV-infected sheep. Furthermore, cyclosporine treatment completely abrogates the difference in kinetics, consistent with a role of the immune response in controlling virus expressing cells.

Reduced levels of reactive oxygen species correlate with inhibition of apoptosis, rise in thioredoxin expression and increased bovine leukemia virus proviral loads

BackgroundBovine Leukemia virus (BLV) is a deltaretrovirus that induces lymphoproliferation and leukemia in ruminants. In ex vivo cultures of B lymphocytes isolated from BLV-infected sheep show that spontaneous apoptosis is reduced. Here, we investigated the involvement of reactive oxygen species (ROS) in this process.ResultsWe demonstrate that (i) the levels of ROS and a major product of oxidative stress (8-OHdG) are reduced, while the thioredoxin antioxidant protein is highly expressed in BLV-infected B lymphocytes, (ii) induction of ROS by valproate (VPA) is pro-apoptotic, (iii) inversely, the scavenging of ROS with N-acetylcysteine inhibits apoptosis, and finally (iv) the levels of ROS inversely correlate with the proviral loads.ConclusionTogether, these observations underline the importance of ROS in the mechanisms of inhibition of apoptosis linked to BLV infection.

Interferon-γ expression associated with suppression of bovine leukemia virus at the early phase of infection in sheep

Immunological control of bovine leukemia virus (BLV)-infection has been reported as dependent on the expression balance of types 1 and 2 cytokines. In this report, mRNA expression of interferon (IFN)-γ and interleukin (IL)-2 (type 1 cytokines), and of IL-4 and IL-10 (type 2 cytokines) were evaluated in concanavalin A-stimulated peripheral blood mononuclear cells (PBMC) from BLV-infected sheep. Despite the same dose of BLV-infection, the extent of viral propagation was markedly different between eight individual sheep by 12 weeks post infection. The virus did not propagate well in three sheep, which showed augmented mRNA expression of IFN-γ, a strong indicator of cell-mediated immunity, immediately after BLV-infection. Among the other five sheep having more than 2% of BLV-infected cells among PBMC at 12 weeks post infection, four sheep developed B-cell leukemia or lymphoma within 2 years after infection. These observations indicate IFN-γ expression may play an important role in the protective mechanism against BLV propagation at the early phase of the infection.

Spleen-Dependent Turnover of CD11b Peripheral Blood B Lymphocytes in Bovine Leukemia Virus-Infected Sheep

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.

Expression of tumor necrosis factor-α in IgM + B-cells from bovine leukemia virus-infected lymphocytotic sheep

Tumor necrosis factor (TNF)-α is thought to be one of the cytokines that account for bovine leukemia virus (BLV)-induced B-cell lymphoproliferative disorder, however, information on TNF-α expression in B-cells is limited. In this study, the expression of TNF-α in IgM + B-cells from BLV-infected sheep with or without lymphocytosis was determined. Freshly isolated IgM + B-cells from three sheep with lymphocytosis constitutively transcribed TNF-α mRNA. Although TNF-α mRNA expression in IgM + B-cells was transiently up-regulated after cell culture, TNF-α mRNA expression was markedly higher in lymphocytotic sheep when compared to that of non-lymphocytotic sheep or uninfected sheep. Expression of membrane-bound TNF-α on IgM + B-cells was also augmented in lymphocytotic sheep. TNF-α expression in lymphocytotic sheep may support the proliferation of B-cells.

Involvement of glutathione as a mechanism of indirect protection against spontaneous ex vivo apoptosis associated with bovine leukemia virus.

Viruses have developed strategies to counteract the apoptotic response of the infected host cells. Modulation of apoptosis is also thought to be a major component of viral persistence and progression to leukemia induced by retroviruses like human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). Here, we analyzed the mechanism of ex vivo apoptosis occurring after isolation of peripheral blood mononuclear cells from BLV-infected sheep. We show that spontaneous apoptosis of ovine B lymphocytes requires at least in part a caspase 8-dependent pathway regardless of viral infection. Cell death is independent of cytotoxic response and does not involve the tumor necrosis factor alpha/NF-kappaB/nitric oxide synthase/cyclooxygenase pathway. In contrast, pharmaceutical depletion of reduced glutathione (namely, gamma-glutamyl-l-cysteinyl-glycine [GSH]) by using ethacrynic acid or 1-pyrrolidinecarbodithioic acid specifically reverts inhibition of spontaneous apoptosis conferred indirectly by protective BLV-conditioned media; inversely, exogenously provided membrane-permeable GSH-monoethyl ester restores cell viability in B lymphocytes of BLV-infected sheep. Most importantly, intracellular GSH levels correlate with virus-associated protection against apoptosis but not with general inhibition of cell death induced by polyclonal activators, such as phorbol esters and ionomycin. Finally, inhibition of apoptosis does not correlate with the activities of GSH peroxidase and GSH reductase. In summary, our data fit into a model in which modulation of the glutathione system is a key event involved in indirect inhibition of apoptosis associated with BLV. These observations could have decisive effects during therapeutic treatment of delta-retroviral pathogenesis.

Ex vivo survival of peripheral blood mononuclear cells in sheep induced by bovine leukemia virus (BLV) mainly occurs in CD5 – B cells that express BLV

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis (EBL). In a previous report, we found that in a sheep model, only CD5 – B cells proliferated clonally, while CD5 + B cells rapidly decreased when the disease progressed to the lymphoma stage. We demonstrate here that, although both CD5 + and CD5 – B cells, but not CD4 + T, CD8 + T and γδTCR +T cells, are protected from spontaneous ex vivo apoptosis in sheep infected with wild-type and a mutant BLV that encodes a mutant Tax D247G protein with elevated trans-activation activity, only CD5 – B cells become the main target for ex vivo survival when the disease proceeds to the persistent lymphocytotic stage, which showed an increased expansion of the CD5 – B cells. In addition, we identified, by four-color flow cytometric analysis, that in CD5 – B cells, the apoptotic rates of cells that expressed wild-type and mutant BLV were greatly decreased compared with those of BLV-negative cells. There was only a slight reduction in the apoptotic rates in BLV-positive cells from CD5 + B cells. In addition, supernatants from peripheral blood mononuclear cell (PBMC) cultures from wild-type- and mutant BLV-infected sheep mainly protected CD5 – B cells from spontaneous apoptosis. Our results suggest that, although BLV can protect both CD5 + and CD5 – B cells from ex vivo apoptosis, the mechanisms accounting for the ex vivo survival between these two B-cell subsets differ. Therefore, it appears that the phenotypic changes in cells that express CD5 at the lymphoma stage could result from a difference in susceptibility to apoptosis in CD5 + and CD5 – B cells in BLV-infected sheep.

Reduced proviral loads during primo-infection of sheep by Bovine Leukemia virus attenuated mutants

BackgroundThe early stages consecutive to infection of sheep (e.g. primo-infection) by Bovine leukemia virus mutants are largely unknown. In order to better understand the mechanisms associated with this period, we aimed at analyzing simultaneously three parameters: B-lymphocytosis, cell proliferation and viral replication.ResultsSheep were experimentally infected either with a wild type BLV provirus or with selected mutants among which: a virus harboring an optimalized LTR promoter with consensus cyclic AMP-responsive elements, two deletants of the R3 or the G4 accessory genes and a fusion-deficient transmembrane recombinant. Seroconversion, as revealed by the onset of an anti-viral antibody response, was detected at 3 to 11 weeks after inoculation. At seroconversion, all sheep exhibited a marked increase in the numbers of circulating B lymphocytes expressing the CD5 and CD11b cluster of differentiation markers and, interestingly, this phenomenon occurred independently of the type of virus. The net increase of the absolute number of B cells was at least partially due to accelerated proliferation as revealed, after intravenous injection of bromodeoxyuridine, by the higher proportion of circulating BrdU+ B lymphocytes. BLV proviral DNA was detected by polymerase chain reaction in the leucocytes of all sheep, as expected. However, at seroconversion, the proviral loads were lower in sheep infected by the attenuated proviruses despite similar levels of B cell lymphocytosis.ConclusionsWe conclude that the proviral loads are not directly linked to the extent of B cell proliferation observed during primo-infection of BLV-infected sheep. We propose a model of opportunistic replication of the virus supported by a general activation process of B lymphocytes.

Latency of viral expression in vivo is not related to CpG methylation in the U3 region and part of the R region of the long terminal repeat of bovine leukemia virus.

Bovine leukemia virus (BLV) is silent in most cells detectable in vivo, and the repression of its expression allows BLV to evade the host's immune response. In this study, we examined whether CpG methylation of DNA might be involved in the regulation of the expression of BLV in vivo. To investigate the effects of CpG methylation on the activity of the long terminal repeat (LTR) of BLV, we measured the transactivation activity of this region after treatment with the CpG methyltransferase SssI by using a luciferase reporter system. The activity of methylated LTR was significantly lower than that of nonmethylated LTR. Therefore, we examined the extent of CpG methylation of the U3 region and part of the R region of the LTR in BLV-infected cattle and in experimentally BLV-infected sheep at various clinical stages by the bisulfite genomic sequencing method. We detected no or minimal CpG methylation at all stages examined in cattle and sheep, and our results indicate that CpG methylation probably does not participate in the silencing of BLV in vivo.

Envelope proteins containing single amino acid substitutions support a structural model of the receptor-binding domain of bovine leukemia virus surface protein.

Functional domains of the strikingly conserved envelope (Env) glycoproteins of bovine leukemia virus (BLV) and its close relative, human T-cell leukemia virus type 1 (HTLV-1), are still being defined. We have used BLV Env protein variants to gain insights into the structure and function of this important determinant of viral infectivity. Each of 23 different single amino acid variants found in cDNA clones of env transcripts present after short-term culture of peripheral blood mononuclear cells from BLV-infected sheep was expressed in COS-1 cells and tested for the ability to mediate cell fusion and to be cleaved to surface (SU) and transmembrane (TM) protein subunits. Of 11 Env variants that failed to induce syncytia or did so poorly, 7 contained changes in amino acids identical or chemically conserved in the HTLV-1 Env protein. These seven included the four variants that showed aberrant proteolytic cleavage and poor cell surface expression, underscoring their importance for Env structure. Ten of 12 variants that retained wild-type syncytium-inducing ability clustered in the N-terminal half of BLV SU, which forms the putative receptor-binding domain (RBD). Several variants in the RBD showed evidence of subtle misfolding, as judged by reduced binding to monoclonal antibodies recognizing conformational epitopes F, G, and H formed by the N terminus of SU. We modeled the BLV RBD by aligning putative structural elements with known elements of the ecotropic Friend murine leukemia virus RBD monomer. All the variant RBD residues but one are exposed on the surface of this BLV model. These variants as well as function-altering, antibody-reactive residues defined by other investigators group on one face of the molecular model. They are strikingly absent from the opposite face, implying that it is likely to face inward in Env complexes. This surface might interact with the C-terminal domain of SU or with an adjacent monomer in the Env oligomer. This location suggests an orientation for the monomer of ecotropic Friend murine leukemia virus RBD.

Increased cell proliferation, but not reduced cell death, induces lymphocytosis in bovine leukemia virus-infected sheep.

Lymphocyte homeostasis is the result of a critical balance between cell proliferation and death. Disruption of this subtle equilibrium can lead to the onset of leukemia, an increase in the number of lymphocytes being potentially due to both of these parameters. The relative importance of cell proliferation vs. apoptosis during pathogenesis induced by the primate T cell lymphotropic viruses and bovine leukemia virus (BLV) has been difficult to assess because of conflicting data from a range of in vitro and ex vivo experimental systems. Here, we aim to resolve this issue by measuring the rates of cell proliferation and death in the BLV-ovine system, an animal model of human T lymphotropic virus (HTLV-1). We use a method based on the i.v. injection of 5-bromodeoxyuridine into BLV-infected sheep. We show that B lymphocytes in BLV(+) asymptomatic sheep proliferate significantly faster than in uninfected controls (average proliferation rate: 0.020 per day vs. 0.011 per day). In contrast, the rates of cell death were not significantly different between aleukemic BLV-infected and control sheep (average death rate 0.089 per day vs. 0.094 per day, respectively). We conclude that the increase in the number of B cells during BLV-induced lymphocytosis results from higher proliferation rates but is not due to a significant decrease in apoptosis, in contrast to data from in vitro (ex vivo) experiments. The imbalance created by the net increase in proliferation in the absence of compensating cell death reveals a complex mechanism of feedback regulation controlling homeostasis in the blood compartment.

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.

Peripheral blood mononuclear cells from sheep infected with a variant of bovine leukemia virus synthesize envelope glycoproteins but fail to induce syncytia in culture

Peripheral blood mononuclear cells (PBMCs) infected with the oncogenic retrovirus bovine leukemia virus (BLV) produce virus when cultured briefly. BLV can be transmitted in cocultures to adherent susceptible cells, which become infected, express viral proteins, and fuse into multinucleated syncytia several days later. PBMCs from 3 of 10 BLV-infected sheep displayed a lifelong deficiency in induction of syncytium formation among indicator cells in culture, although large numbers of PBMCs synthesized viral transcripts or capsid protein. Since the infected, syncytium-deficient PBMCs were > or = 97% B cells, the deficiency could not be attributed to altered host cell tropism. The syncytium-deficient phenotype was recapitulated in newly infected sheep, demonstrating that this property is regulated by the viral genotype. The alteration in the BLV genome delayed but did not prohibit the establishment of BLV infection in vivo. Envelope glycoproteins were synthesized in syncytium-deficient PBMCs, translocated to the cell surface, and incorporated into virions. However, monoclonal antibodies specific for the BLV surface glycoprotein did not stain fixed PBMCs of the syncytium-deficient phenotype. Moreover, an animal with syncytium-deficient PBMCs had lower titers of neutralizing antibodies throughout the first 5 years of infection than an animal with similar numbers of infected PBMCs of the syncytium-inducing phenotype. The syncytium-deficient variant productively infected indicator cells at greatly reduced efficiency, showing that the alteration affects an early step in viral entry or replication. These results suggest that the alteration maps in the env gene or in a gene whose product affects the maturation or conformation, and consequently the function, of the envelope protein complex.

Proliferating cell nuclear antigen expression in sheep infected with bovine leukemia virus

Proliferating cell nuclear antigen (PCNA) or cyclin (C), a major nuclear protein, has been shown to be associated with human leukemia and malignancies. PCNA protein was quantitated in this study, in lymphocytes from bovine leukemia virus (BLV) infected and non-infected sheep, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and silver staining. The PCNA mean levels in lymphocytes of BLV-infected sheep (27 months post-infection) was significantly ( P<0.001) higher than in the lymphocytes of the non-infected sheep. The mean of PCNA levels in lymphocytes of sheep, 21 days after BLV infection, showed a two-fold increase compared with the non-infected sheep. Phytohemagglutinin (PHA) (3 days) treatment of lymphocytes from the non-infected and from the BLV-infected sheep resulted in a significant ( P<0.01) increase in the mean of PCNA levels only in the non-infected sheep. The mean lymphocyte counts of the BLV-infected sheep were not significantly different from the mean counts of the non-infected sheep at the time of lymphocyte protein analysis. Thus, these findings showed, similar to human leukemia and malignancies, that high levels of PCNA were found in lymphocytes from BLV-infected sheep compared with those from the non-infected sheep, and this was independent of high cell count. Our results also suggest that PCNA protein may play a role in the process of lymphoid transformation as a result of BLV infection of sheep.

Bovine Leukemia Virus Induces CD5 - B Cell Lymphoma in Sheep Despite Temporarily Increasing CD5 + B Cells in Asymptomatic Stage

To investigate bovine leukemia virus (BLV)-induced leukemogenesis, we infected sheep with BLV and used flow-cytometric and immunohistological analysis to characterize the phenotypic alterations in lymphocytes from peripheral blood and lymph nodes taken from the animals with lymphoma at various stages. In sheep at the asymptomatic stage, depending on the extent of progression of the disease, the proportions of CD2 +-, CD4 +-, CD8 +-, and γδTCR +-T cells that coexpressed CD5 decreased, but CD5 +slgM + cells as well as CD5 -slgM + cells increased for a period. The number of CD5 + B cells, however, rapidly decreased in the lymphoma stage. On the other hand, neoplastic lymphocytes appeared to be a monoclonal population derived from a single cell with surface phenotypes of slgM +, B-cell-specific molecule B2 +, major histocompatibility complex (MHC) class II +, OvCD5 -, OvCD2 -, OvCD4 -, OvCD8 -, γδTCR -, which suggests that only CD5 - B cells proliferate clonally when the disease proceeds to the lymphoma stage. Thus, rapid decrease of CD5 + B cells may be used as a marker of lymphoma stage. To identify the BLV provirus in the CD5 - B and CD5 + B cells throughout the course of disease, each fraction of CD5 - B and CD5 + B cell was sorted from the peripheral blood by flow cytometry and nested double polymerase chain reaction was performed. In BLV-infected but healthy sheep, BLV integrated both CD5 - B and CD5 + B cells. In lymphoma, however, BLV provirus was detected only in CD5 - B cells but not in CD5 + B cells. Therefore it appears that a disappearance of BLV-infected CD5 + cells is one of the critical events leading to CD5 - B cell lymphoma in sheep. This is in contrast to the BLV-induced lymphoma in cattle which shows CD5 + phenotype.

Immunopathologic study and characterization of the phenotype of transformed cells in sheep with bovine leukemia virus-induced lymphosarcoma

Summary We used monoclonal antibodies and immunohistologic examination of lymph nodes, to elucidate the pathogenesis of lymphosarcoma induced by infection with bovine leukemia virus (blv). The superficial cervical lymph nodes from 3 blv-infected but apparently healthy sheep and 5 sheep with fullblown lymphosarcoma were examined. We also investigated the integration of bovine leukemia provirus by use of Southern blotting. In lymph nodes from sheep lacking clinical signs of infection, in which the provirus had been integrated at multiple sites in the genome, many large hypertrophic follicles were observed in the cortex. These follicles had germinal centers consisting of CD4+T cells and B cells that expressed surface IgM (slgM) and major histocompatibility complex (mhc) class-II antigens, but not B cell-specific B2 molecule. The percentage of CD4+T cells in the cortex was significantly (P &lt; 0.05) higher than that of the controls and sheep with lymphosarcoma. In all sheep with lymphosarcoma, the lymph nodes were completely destroyed by proliferating neoplastic cells, and in addition, small atrophic follicles, which consisted of normal B-cell marker-positive cells, were seen near the trabecula and the subcapsule. In these instances, neoplastic cells appeared to be a monoclonal population derived from a single CD5− B-cell lineage and to be classified as 2 types, CD5−CD4−CD8−B2+MHC class-II+sIgM+ and CD5−CD4−CD8−B2+mhc class-II+sIgM−. Moreover, CD8+T cells infiltrated diffusely throughout the tumorous lymph nodes apart from the atrophic follicles, and CD4+T cells were observed around atrophic follicles. Both types of T cells were small-size, normal lymphocytes with round and noncleaved nuclei, and were apparently non-neoplastic cells. In fact, after separation by use of a panning method, it seems that, in blood mononuclear cells from BLV-infected sheep without clinical signs of infection, but in lymphosarcomatous stages, the proviral genome was integrated only in B cells and not in T cells. Thus, we conclude that the host's immune response may be still maintained at a lymphosarcomatous stage.

T-cell responses to highly conserved CD4 and CD8 epitopes on the outer membrane protein of bovine leukemia virus: relevance to vaccine development

Bovine leukemia virus (BLV) is a retrovirus that infects cattle and sheep and may provide a model for studying human leukemia. Cell-mediated immune mechanisms may play a major role in protection against BLV infection. We describe here for the first time the identification of proliferative (CD4) and cytotoxic T-lymphocyte (CD8) epitopes of the gp51 envelope (env) protein of BLV. This protein and a recombinant form expressed by a vaccinia virus construct have been shown to be potential vaccine candidates. A complete series of overlapping peptides, 20 amino acids in length, was prepared to identify epitopes from gp51. These peptides were tested for the ability to elicit peripheral blood lymphocyte proliferation and cytotoxic T-lymphocyte responses in infected and uninfected cattle and sheep. Peptides 51-70 and 61-80 produced a proliferative response in lymphocytes from only uninfected animals (both sheep and cattle), and this was shown by T-cell subset deletion to be a CD4-mediated response. Seven BLV-infected sheep did not show a response to either peptide. Cytotoxic T-lymphocyte activity, however, was associated only with peptides 121-140 and 131-150. In this case, the response was demonstrated to be CD8 dependent and was found only in BLV-infected animals (sheep). Knowledge of the location of these T-cell recognition domains will complement data available on B-cell epitopes in gp51 and may be useful in the design of a subunit vaccine.

Activation of bovine leukemia virus transcription in lymphocytes from infected sheep: rapid transition through early to late gene expression.

Bovine leukemia virus (BLV) expression is mostly silent in peripheral blood mononuclear cells (PBMCs) of infected animals. However, when infected cells are cultured, they are stimulated to produce virus. We studied viral transcription in PBMCs taken from BLV-infected sheep because the pattern of transcriptional activation in these cells should closely mimic activation of virus expression within mononuclear cells in vivo. BLV transcription was activated as early as 30 min after PBMCs were cultured. Expression was characterized by early and late stages, each distinguished by a unique pattern of cytoplasmic RNAs. In early expression, cytoplasmic viral RNA was exclusively the doubly spliced tax/rex transcript, although all transcripts were present in the nucleus. Early expression gave way rapidly to late expression, in which all viral transcripts accumulated in the cytoplasm. The polyclonal B-cell activator lipopolysaccharide increased the amount of viral RNA by at least twofold but did not alter the pattern of transcription. The transition from early to late expression required new protein synthesis and was blocked by the inhibitor cycloheximide. This requirement reflects the essential role of the viral Rex protein in the transition, but synthesis of cellular factors may be required as well. These results provide the first demonstration of staged viral expression in lymphocytes naturally infected by either BLV or the closely related human T-cell leukemia virus (HTLV) and validate the model of BLV and HTLV gene expression that previously was derived from transfection experiments performed mainly in nonlymphoid cells.

In vivo transcription of the bovine leukemia virus tax/rex region in normal and neoplastic lymphocytes of cattle and sheep

Expression of bovine leukemia virus (BLV) has been considered to be blocked at the transcriptional level in vivo, since viral RNA species are not readily detected in freshly isolated leukocytes from BLV-infected animals. However, the presence of a persistent antiviral antibody response in infected animals suggests that some degree of virus expression must occur in vivo. The purpose of this study was to determine whether BLV RNA species could be detected by using the polymerase chain reaction in normal or neoplastic lymphoid cells freshly isolated from naturally or experimentally BLV-infected cattle and sheep, respectively. Primers designed to detect a 2.1-kb doubly spliced BLV tax/rex-specific mRNA were used to amplify cDNA copies of RNA derived from infected animals. The amplified viral product was then detected with a radiolabeled BLV tax/rex-specific probe. BLV-specific RNA was detected readily in freshly isolated peripheral blood leukocytes derived from BLV-seropositive cattle or sheep with persistent lymphocytosis and less readily in peripheral blood leukocytes from BLV-seropositive but hematologically normal animals. BLV-specific RNA was also detected in fresh samples of BLV-induced lymphosarcomas. Normal and neoplastic lymphoid cells from BLV-seronegative animals were uniformly negative under similar conditions. These primers also amplified the same viral product from genomic DNA derived from BLV-seropositive animals, providing further evidence for in vivo transcription and suggesting that BLV RNA-dependent DNA polymerase is capable of reverse transcribing the 2.1-kb mRNA in vivo. The demonstration of transcriptional products of BLV in vivo proves that viral latency in BLV infection is incomplete.