- Open Access
A detailed molecular analysis of complete Bovine Leukemia Virus genomes isolated from B-cell lymphosarcomas
© Moratorio et al.; licensee BioMed Central Ltd. 2013
Received: 30 August 2012
Accepted: 30 January 2013
Published: 18 March 2013
It is widely accepted that the majority of cancers result from multiple cellular events leading to malignancy after a prolonged period of clinical latency, and that the immune system plays a critical role in the control of cancer progression. Bovine leukemia virus (BLV) is an oncogenic member of the Retroviridae family. Complete genomic sequences of BLV strains isolated from peripheral blood mononuclear cells (PBMC) from cattle have been previously reported. However, a detailed characterization of the complete genome of BLV strains directly isolated from bovine tumors is much needed in order to contribute to the understanding of the mechanisms of leukemogenesis induced by BLV in cattle. In this study, we performed a molecular characterization of BLV complete genomes from bovine B-cell lymphosarcoma isolates. A nucleotide substitution was found in the glucocorticoid response element (GRE) site of the 5' long terminal repeat (5'LTR) of the BLV isolates. All amino acid substitutions in Tax previously found to be related to stimulate high transcriptional activity of 5'LTR were not found in these studies. Amino acid substitutions were found in the nucleocapsid, gp51 and G4 proteins. Premature stop-codons in R3 were observed. Few mutations or amino acid substitutions may be needed to allow BLV provirus to achieve silencing. Substitutions that favor suppression of viral expression in malignant B cells might be a strategy to circumvent effective immune attack.
Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic member of the Retroviridae family that infects cattle worldwide and is the causative agent of enzootic bovine leukosis (EBL), a neoplastic proliferation of B cells [1, 2]. BLV infection is characterized by a long period of viral latency and by the absence of viremia. This is thought to be related to the transcriptional repression of viral expression in vivo . Latency is likely a viral strategy to evade the host immune response, thereby allowing tumor development [4, 5]. In fact, B lymphocytes harboring an integrated provirus do not produce detectable levels of viral RNA or proteins . Nevertheless, when these cells are isolated and cultured in vitro, a marked increase in viral transcription occurs, suggesting that the provirus is maintained at a repressed stage in vivo .
Regarding genome organization, as in all retroviruses, BLV has the gag, pro, pol, env structural genes (from 5′ to 3′ of the genome) required for the production of infectious virions . In addition to these genes, the BLV genome contains an X region located between the env gene and the 3′ long terminal repeat (3′-LTR) , as also observed in other Deltaretroviruses . This region contains the open reading frames of four regulatory proteins: the transactivator protein, Tax ; the Rex protein, which stabilizes and allows exportation through the cytoplasm of viral RNA  and two accessory proteins R3 and G4 whose small open reading frames (ORF) are located in the region between the env gene and the tax/rex genes . Deletion of R3 and G4 genes of BLV in an infectious and tumorigenic BLV molecular clone induced loss of the leukomogenic phenotype and G4 exhibited oncogenic potential both in vivo and in vitro [14, 15].
The BLV transcriptional promoter is located in the 5′long terminal repeat (5′-LTR) and is composed of the U3, R and U5 regions. Gene expression is induced at the transcriptional level by the virus-encoded transactivator Tax .
Few complete genomic sequences of BLV strains are available in the databases. These sequences are from different sources: peripheral blood mononuclear cells (PBMC) , tumor cells, experimentally infected sheep, and cell lines (FLK). The degree of genetic variation among these strains and those directly isolated from bovine B-cell lymphosarcomas remains unknown. For this reason, and in order to contribute to the understanding of the mechanisms of leukemogenesis induced by BLV, we performed a detailed characterization of the complete genome of three BLV isolates from B-cell lymphosarcomas of three cows from different farms, and we compared them with all available and corresponding full length sequences from BLV isolates from other sources.
Materials and methods
Lymphosarcoma samples were obtained by certified veterinary doctors following appropriate ethical guidelines from national and international veterinary associations. The project was also read and approved by Institut Pasteur-Montevideo, Uruguay.
Lymphosarcoma samples were obtained from three dairy cows proven to be infected with BLV by PCR and ELISA (VMRD Inc., Pullman, WA, USA).
DNA extraction and PCR amplification
DNA samples were extracted from lymphosarcoma tissue and FLK cells (as a control), using the QiAmp DNA Blood Mini kit from QIAGEN, according to the instructions supplied by the manufacturer. PCR amplification of overlapping genome fragments covering the complete genome of BLV was achieved using Phusion DNA Polymerase (New England BioLabs) and specific primers designed for this study (synthesized by Integrated DNA Technologies, Leuven, Belgium and shown in Additional file 1). The location of each amplicon is shown in Additional file 2. Reagents for PCR were from New England BioLabs. The final reaction mixture (50 μL) contained 1x HF buffer, 200 μM dNTP, 200 nM of each primer, and 1 U Taq polymerase. The cycle for the PCR amplification were as follows: 98°C for 30 s, then 30 cycles of denaturation at 98°C for 10 s, annealing at 55–65°C for 30 s, and extension at 72°C for 1–3 min, followed by a final extension at 72°C for 10 min. The PCR reactions were carried out using an Eppendorf Mastercycler Gradient PCR Thermal Cycler.
Amplicon purification and cloning
Amplicons were resolved by 1% agarose gel electrophoresis, stained with ethidium bromide and purified using QIAquick PCR Purification Kit from QIAGEN, according to instructions from the manufacturers, and cloned into pGEM T- Easy vector (Promega). Electrocompetent XL1-Blue bacteria were transformed by colonies and were expanded and small-scale plasmid purification was performed using the GFX DNA purification kit (GE Healthcare, Piscataway, NJ, USA).
Both strands of purified plasmids were sequenced in order to avoid discrepancies by using specific and universal T7 or SP6 primers and the Big Dye DNA sequencing kit (Perkin-Elmer) on a 373 DNA sequencer apparatus (Perkin-Elmer). Complete genome sequences were obtained from B-cell lymphosarcomas and deposited in the EMBL database under accession numbers EMBL:HE967301 to EMBL:HE967303 (LS1to LS3). Complete genome sequences were obtained for all available and comparable BLV strains by using All-round Retrieval of Sequence and Annotation (ARSA) at the DNA Data Bank of Japan (DDBJ) .
Sequences were aligned using the CLUSTAL W program .
Protein sequences were obtained by means of in silico translation of nucleotide to amino acid sequences. This was done by using software from the MEGA program .
Results and discussion
Comparison of the 5′-LTR genome region of BLV strains isolated from lymphosarcomas and other origins
Comparison of the 5′-LTR genomic sequences of the three BLV lymphosarcoma isolates (LSI) with all available complete BLV genome sequences, revealed that this genome region is highly conserved (Figure 1). The only significant difference between LSI and those isolated from other cell types, e.g. PBMC or FLK cells, is a base substitution found at position 150 (G to A) in the third enhancer element of this region, at the GRE binding site (Figure 1). It has been previously found that GRE confers responsiveness to glucocorticoids such as dexamethasone in the presence of the Tax transactivator . However, in the absence of Tax, mutation of the GRE significantly decreases basal LTR activity as shown in reporter-based assays . This raises the possibility that this substitution may have allowed a better silencing of viral transcription in the lymphosarcoma strains, as a strategy to avoid recognition by the host immune response .
Comparison of deduced amino acid sequences from structural proteins of BLV LSI with those of other origins
BLV protease (PR) is an aspartic protease with a functional activity involved in gag processing and thus in virion maturation. Previous work proposed a molecular model for BLV PR as well as its substrate specificity, cleavage type sites and inhibitor sensitivity . The comparison of amino acid sequences of PR of BLV LSI with all other sequenced BLV isolates examined in this study is shown in Figure 2B. Only one amino acid substitution (V165I) was found among the BLV lymphosarcoma isolates and is not related to sites previously reported to be involved in BLV PR function via main-chain atoms of peptide substrates or residues predicted to form cleavage subsites [32, 33]. Two substitutions can be observed at positions 37–38 in lymphosarcoma BLV isolate LS1, as compared to other genomic sequences including LS2 and 3 isolates (see Figure 2B).
This substitution could involve important structural changes, but unfortunately, the structure of BLV polymerase as well as other related Deltaretroviruses, like HTLV-1, is currently unknown.
Further studies will be needed to establish if these substitutions can affect polymerase fidelity or processivity.
The Env protein complex is composed of two component subunits: gp51 surface (SU, N- terminal portion) and gp30 transmembrane (TM, C-terminal portion), which remain associated as a functional trimer with three SU subunits linked by disulphide bonds to a spike of three TM subunits . The gp51protein recognizes and binds to cellular receptors, thereby initiating conformational changes that lead to fusion of viral and cellular membranes by gp30 oligomers .
Comparison of deduced amino acid sequences from non-structural proteins of BLV LSI and other origins
Previous studies revealed that silencing is critical for tumor progression and distinct genetic and epigenetic mechanisms were identified for complete suppression of BLV Tax expression.
Conservation of sites involved in suppression of viral expression may be an important factor for the uncontrolled proliferation of BLV-infected tumor cells .
The Rex proteins of Deltaretroviruses act to facilitate the export of intron-containing viral RNA . The Rex proteins shuttle between nucleus and cytoplasm using the nuclear localization signal (NLS) and nuclear export signal (NES) (see Figure 5B). No significant substitutions were found in Rex proteins of all BLV strains enrolled in this study.
No amino acid substitutions were found in the arginine-rich α-helix of G4 protein of the previously sequenced BLV isolates examined in this work (see Figure 6A). Nevertheless, an amino acid substitution (A29V) can be observed in G4 of all BLV LSI.
Interestingly, premature stop codons were observed in R3 of two of the three LS BLV isolates (Figure 6B). Previous studies on BLV infection using sheep provide insight on the molecular genetic and epigenetic modulation of viral expression . These studies show that the deletion of the region that expands from the end of the env gene to the splice acceptor site of the tax/rex mRNA does not impair infectivity . These sequences correspond to the third and second exons of R3 and G4, respectively, revealing that these sequences may not be essential for infectivity in vivo. Although previous studies have shown that deletions in R3/G4 interferes with the efficiency of BLV propagation and restricts pathogenesis [14, 15, 46, 49], another study has shown that one out of 20 sheep infected with a R3/G4 mutant developed a lymphoma after 7.5 years of latency, suggesting that the deleted sequences may not be strictly required for pathogenesis . Further studies will be needed to address the biological significance of these findings in studies using the cow as a model for BLV infection.
In summary, although the genome of BLV is highly conserved in our isolates and in isolates from other sources previously described, variations can be observed in some genome regions. It is thought that silencing of viral expression is a multi-step process leading to the uncontrolled growth of a transformed B-cell clone and the onset of disease  and is critical for tumor progression and proliferation of BLV-infected tumor cells , as well as escaping recognition by the host immune response . In that sense, the substitution found in the GRE site of the 5′LTR of all BLV strains isolated from the lymphosarcomas might contribute to these factors, since previous studies have shown that substitutions in GRE site significantly reduces basal LTR transcription activity  (see Figure 1). Moreover, all amino acid substitutions in Tax previously found to be related to stimulate high transcriptional activity of 5′LTR were not found in this study (see Figure 5A). Genetic and epigenetic mechanisms have been recently proposed for BLV suppression of viral gene expression . The results of the present report, using full-length genome sequences, suggest that point mutations along the whole genome may also be needed to allow BLV provirus to achieve silencing.
We acknowledge support from Programa de Desarrollo de Ciencias Básicas (PEDECIBA), Agencia Nacional de Investigación e Innovación (ANII) and Instituto Nacional de Investigación Agropecuaria (INIA), Uruguay. We acknowledge anonymous reviewer’s for important suggestions and contributions to improve the quality of this work.
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