A Mutant Form of the Tax Protein of Bovine Leukemia Virus (BLV), with Enhanced Transactivation Activity, Increases Expression and Propagation of BLV In Vitro but Not In Vivo

Abstract

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.Bovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), causes enzootic bovine leukosis, a disease characterized by a very extended course that often involves persistent lymphocytosis and culminates in B-cell lymphoma (5). Under experimental conditions, sheep can easily be infected with BLV and tend to develop B-cell leukemia/lymphoma at higher frequencies and after shorter latent periods than cattle (3,8).In addition to the involvement of the classical structural genes of retroviruses, BLV has a region, designated pX and located between the env gene and the 3Ј long terminal repeat (LTR), that contains at least seven open reading frames (21). Two well-characterized proteins encoded by the pX region, Tax and Rex, are involved in regulation of the expression and replication of BLV (6, 7). The Tax protein acts on a triplicate 21-bp enhancer motif, known as the Tax-responsive element, in the U3 region of the 5Ј LTR and stimulates transactivation of the viral genome (6, 29). The Tax-responsive element consists of a sequence that resembles a cyclic AMP response element, and it has been suggested that Tax binds indirectly to this element through a member of the CREB/ATF family (1, 2, 4, 31). Furthermore, the Tax protein can cooperate with the Ha-Ras oncoprotein to induce full transformation of primary rat embryo fibroblasts (30), an observation that suggests that Tax not only activates the replication of BLV but also contributes to the induction of lymphoma. The Tax protein o...

Fate of Premalignant Clones during the Asymptomatic Phase Preceding Lymphoid Malignancy

“…Amplification conditions were 958C for 8 minutes to activate the polymerase, followed by 55 cycles at 958C for 10 seconds, 608C for 10 seconds, and 728C for 10 seconds. In order to standardize the amount of DNA subjected to quantification, we used the sheep h-globin gene as an internal standard, as described by Tajima et al (8). The standard curve for h-globin was generated using DNA extracted from BLV-negative sheep blood cells.…”

Cell dynamics and immune response to BLV infection: a unifying model

“…Why would a viral antigen-specific B cell be preferentially infected by the virus? We therefore favor a model in which the virus plays an active role by continuously expressing viral proteins, like the Tax oncogene, able to promote cell proliferation and transformation (26,49,(65)(66)(67). Tax expression could be permanent providing that cells escape from immune response, which is a rare event, or initiated indirectly via cellular activation.…”

“…And: what is the driving force of the clonal expansion process? Based on the extensively described oncogenic properties of Tax (26,(65)(66)(67), our tenet is that this virally encoded protein triggers cell proliferation. However, the selective growth advantage of the infected cells could also possibly be provided by a non-peptidic viral factor, such as for instance microRNAs (68).…”

Tumor necrosis factor-alpha genetic polymorphism may contribute to progression of bovine leukemia virus-infection

“…Purified genomic DNA concentration was measured by optical density (OD) at 260 nm and stored at 4°C until use. BLV infection was tested by the agar gel immuno-diffusion assay using the BLV env glycoprotein gp51 as antigen, and was further confirmed by real-time PCR, using primer pair BLV-LTR256 (5'-GAG CTC TCT TGC TCC CGA GAC-3') and BLV-LTR453 (5'-GAA ACA AAC GCG GGT GCA AGC CAG-3'), to amplify the BLV long terminal region (LTR) using a LightCycler TM (Roche Diagnostics, Mannheim, Germany) as described previously [14]. BLV-infected cells were quantified based on viral genome amplification by real-time PCR as described previously [14].…”