Isolation of a bovine plasma fibronectin-containing complex which inhibits the expression of bovine leukemia virus p24

Bovine leukemia virus (BLV) is a deltaretrovirus that infects cattle worldwide. In agriculturally intensive regions, approximately 30% of dairy cows are BLV infected. Like the human T-cell leukemia virus (HTLV), there is a lengthy period of viral quiescence after initial infection with BLV. Unlike HTLV, BLV resides predominantly in B cells. Lymphoma is observed in less than 10% of BLV-infected adult cattle. Although viremia is undetectable in vivo, BLV-infected peripheral blood mononuclear cells readily become productive when cultured in vitro. Productivity is markedly diminished when cultures are supplemented with bovine plasma. This inhibitory activity of bovine plasma has been attributed to the "plasma blocking factor" (PBF). Here, we describe the purification of a PBF whose activity was resistant to heating to 65 degrees C for 10 min and was attributable to a fibronectin-containing complex of approximately 320 kDa under nonreducing conditions. By use of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry), a protein with a size of 220 kDa and a pI of 5.4 was identified as a member of the fibronectin group of molecules. Both the purified protein and the commercially available bovine fibronectin inhibited BLV production in naturally infected peripheral blood mononuclear cells, although the fibronectin was less biologically active.     

Complete Bovine Leukemia Virus (BLV) Provirus Is Conserved in BLV-Infected Cattle throughout the Course of B-Cell Lymphosarcoma Development

Bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) belong to the same subfamily of oncoviruses. Defective HTLV-1 proviral genomes have been found in more than half of all patients with adult T-cell leukemia examined. We have characterized the genomic structure of integrated BLV proviruses in peripheral blood lymphocytes and tumor tissue taken from animals with lymphomas at various stages. Genomic Southern hybridization with SacI, which generates two major fragments of BLV proviral DNA, yielded only bands that corresponded to a full-size provirus in all of 23 cattle at the lymphoma stage and in 7 BLV-infected but healthy cattle. Long PCR with primers located in long terminal repeats clearly demonstrated that almost the complete provirus was retained in all of 27 cattle with lymphomas and in 19 infected but healthy cattle. However, in addition to a PCR product that corresponded to a full-size provirus, a fragment shorter than that of the complete virus was produced in only one of the 27 animals with lymphomas. Moreover, when we performed conventional PCR with a variety of primers that spanned the entire BLV genome to detect even small defects, PCR products were produced that specifically covered the entire BLV genome in all of the 40 BLV-infected cattle tested. Therefore, it appears that at least one copy of the full-length BLV proviral genome was maintained in each animal throughout the course of the disease and, in addition, that either large or small deletions of proviral genomes may be very rare events in BLV-infected cattle.     

Reduced bovine leukaemia virus proviral load in genetically resistant cattle

The bovine leukaemia virus (BLV) is an exogenous retrovirus that is closely related to the human T cell leukaemia viruses. Genetic resistance and susceptibility to persistent lymphocytosis (PL), an advanced subclinical stage of infection characterized by a polyclonal expansion of the infected B cell population, have been mapped to structural motifs in bovine MHC DRB3 (class II) alleles. To determine whether alleles of DRB3 influence the number of BLV-infected B cells in peripheral blood, seven pairs of Holstein cows naturally infected with BLV were matched on the basis of DRB3 genotype (resistance or susceptibility to PL), age, and year of seroconversion. Flow cytometry was used to separate B cell populations that then were tested for the presence of provirus by a single-cell PCR methodology. Animals with the PL-resistance associated DRB3.2*11 allele had significantly fewer BLV-infected B cells than did age- and seroconversion-matched cows with DRB3 alleles associated with susceptibility to PL. Our results demonstrate that DRB3 or a closely linked gene may play a direct role in controlling the number of BLV-infected peripheral B cells in vivo . Association of MHC class II alleles with resistance to disease progression in cattle naturally infected with BLV provides a unique immunogenetic model for the study of host resistance to human and other animal retroviral infections.     

An improved syncytia infectivity assay for the bovine leukemia virus.

Several factors that influence the sensitivity of the syncytia infectivity assay for the bovine leukemia virus (BLV) and BLV-infected lymphocytes have been examined. The use of early-passage indicator bovine embryonic spleen (BESP) cells and their pretreatment with diethylamino-ethyl-dextran (DEAE-D) was essential for optimal sensitivity. Polybrene was less effective than DEAE-D. The combination of DEAE-D and polybrene was more effective than DEAE-D alone when BLV-infected leukocytes were used as the inoculum, but not when the inoculum was a cell-free BLV preparation. Using BESP cell passages 4 to 11 as indicators, reproducible titers were obtained when aliquots of the same virus stock were assayed at different times after freezer storage. When assaying peripheral blood lymphocytes from infected cattle, optimal syncytia responses were observed consistently by inoculating 5 X 10(6) viable lymphocytes per 60-mm Falcon dish. Centrifugation of peripheral blood leukocytes from BLV-infected cattle in discontinuous bovine serum albumin gradients can be used to separate a subpopulation of infected lymphocytes. Use of this subpopulation as the inoculum, rather than unseparated buffy-coat leukocytes, greatly increases the sensitivity of the syncytia infectivity assay.     

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

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

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.     

An improved syncytia infectivity assay for the bovine leukemia virus

Summary Several factors that influence the sensitivity of the syncytia infectivity assay for the bovine leukemia virus (BLV) and BLV-infected lymphocytes have been examined. The use of early-passage indicator bovine embryonic spleen (BESP) cells and their pretreatment with diethylamino-ethyl-dextran (DEAE-D) was essential for optimal sensitivity. Polybrene was less effective than DEAE-D. The combination of DEAE-D and polybrene was more effective than DEAE-D alone when BLV-infected leukocytes were used as the inoculum, but not when the inoculum was a cell-free BLV preparation. Using BESP cell passages 4 to 11 as indicators, reproducible titers were obtained when aliquots of the same virus stock were assayed at different times after freezer storage. When assaying peripheral blood lymphocytes from infected cattle, optimal syncytia responses were observed consistently by inoculating 5×10⁶ viable lymphocytes per 60-mm Falcon dish. Centrifugation of peripheral blood leukocytes from BLV-infected cattle in discontinuous bovine serum albumin gradients can be used to separate a subpopulation of infected lymphocytes. Use of this subpopulation as the inoculum, rather than unseparated buffy-coat leukocytes, greatly increases the sensitivity of the syncytia infectivity assay. This work was supported in part by USPHS Grant 1-PO 1-CA-14193-03, Pennsylvania Department of Agriculture Grant ME4, and USDA Cooperative Agreement 12-14-100-10, 675 (45).     

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.     

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.     

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.     

Association between BoLA and subclinical bovine leukemia virus infection in a herd of Holstein-Friesian cows

The role of the bovine major histocompatibility system (BoLA) in subclinical bovine leukemia virus (BLV) infection was investigated in a herd of Holstein-Friesian cows (n=240). The BoLA W8.1 allele was negatively associated with the presence of antibodies to the major BLV envelope glycoprotein, BLV-gp51 (corrected P<0.001, relative risk =0.31). These results suggest that a BoLA-linked gene(s) may influence the early spread of BLV infection. Since B cells are the primary target of BLV infection, we then determined the relationship between BoLA-A locus phenotypes and B-cell numbers in peripheral blood of seropositive and seronegative cows. There were no significant differences between BoLA-A alleles for any hematological parameter in seronegative cows. Seropositive cows with the W12.1 allele had significantly greater absolute numbers of lymphocytes per microliter and B cells per microliter than did seropositive cows with other BoLA-A phenotypes (P<0.01, respectively). The average effect associated with the W12.1 allele in BLV-infected cows was an increase of 2010 B cells per microliter of whole blood relative to BLV-infected cows with other BoLA-A phenotypes. These results demonstrate that susceptibility to the polyclonal expansion of BLV-infected B lymphocytes is associated with the W12.1 allele in Holstein-Friesian cattle. Compared with results of a previous study in a herd of Shorthorn cattle, it appears that resistance and susceptibility to subclinical progression of BLV infection are associated with different BoLA-A locus alleles in different cattle breeds.Abbreviations used in this paper AGID agar gel immunodiffusion - BLV bovine leukemia virus - BoLA bovine lymphocyte antigen - EBL enzootic bovine leukosis - HLA human leukocyte antigen - MHC major histocompatibility complex - PL persistent lymphocytosis     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

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

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

Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown.