Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep

Abstract

Background: During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. In the Bovine Leukemia Virus-induced ovine leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion. However, whether proviral expression in tumors is completely suppressed in vivo was not conclusively demonstrated. Therefore, we studied viral expression in two selected experimentally-infected sheep, the virus or the disease of which had features that made it possible to distinguish tumor cells from their nontransformed counterparts.

Results: In the first animal, we observed the emergence of a genetically modified provirus simultaneously with leukemia onset. We found a Tax-mutated (TaxK303) replication-deficient provirus in the malignant B-cell clone while functional provirus (TaxE303) had been consistently monitored over the 17-month aleukemic period. In the second case, both non-transformed and transformed BLV-infected cells were present at the same time, but at distinct sites. While there was potentially-active provirus in the non-leukemic blood B-cell population, as demonstrated by ex-vivo culture and injection into naïve sheep, virus expression was completely suppressed in the malignant B-cells isolated from the lymphoid tumors despite the absence of genetic alterations in the proviral genome. These observations suggest that silencing of viral genes, including the oncoprotein Tax, is associated with tumor onset.

Conclusion: Our findings suggest that silencing is critical for tumor progression and identify two distinct mechanisms-genetic and epigenetic-involved in the complete suppression of virus and Tax expression. We demonstrate that, in contrast to systems that require sustained oncogene expression, the major viral transforming protein Tax can be turned-off without reversing the transformed phenotype. We propose that suppression of viral gene expression is a contributory factor in the impairment of immune surveillance and the uncontrolled proliferation of the BLV-infected tumor cell.

Figure 1

Follow-up of sheep S2531: silencing occurssimultaneously with the onset of leukemia. (A) Blood samples were collected from S2531 at regular time intervals over a 18-month period from the time of inoculation to the leukemic stage and examined for several parameters. WBC counts per mm³ are indicated. Provirus load and integration were examined by Southern blot hybridization of SacI- and EcoRI-digests respectively, showing increasing provirus load and the progression from polyclonal to monoclonal integration as leukemia develops. The nucleotide sequence of the 3' end of the proviral tax DNA is illustrated by a polyacrylamide gel autoradiography of dideoxynucleotide sequenced PCR-amplified DNA. Boxes highlight nucleotides at positions 8149, 8150 and 8151 of the BLV sequence [29]. Arrows indicate the nucleotide identified at position 8149: a G at pre-leukemic stages (yellow arrow); a G to A transition at the time of the first documented WBC increase (17-month post-infection, red arrow). The resulting amino acid at position 303 of the corresponding Tax proteins is shown below. The transactivation potential of the putative S2531 proviral Tax proteins were examined in a luciferase reporter assay following co-transfection of HeLa cells with the pSGTax₂₅₃₁ expression vectors containing tax sequences cloned from S2531 PBMCs collected at different times post-infection and the reporter plasmid pLTR-Luc as detailed in B. "+" indicates a luciferase activity equivalent to that resulting from transfection with the wild-type pSGTax; "-" indicates the background level activity similar to that obtained when the empty expression vector pSG5 is co-transfected with pLTR-Luc. (B) Luciferase assay reflecting the transactivation potential of a selection of four S2531-derived tax sequences. Each pSGTax₂₅₃₁ construct containing the different S2531-derived tax sequences downstream of the CMV promoter was used in HeLa co-transfection with pLTR-Luc which expresses the firefly luciferase under the control of the BLV-LTR promoter. Luciferase activities were measured in cell lysates 42 h posttransfection and were normalized to protein concentrations as previously described [19]. Results are represented as histograms indicating basal luciferase activities (arbitrary units). pSGTax_2531–6 and pSGTax_2531–14 contain sequences amplified from PBMCs isolated during the aleukemic stage, 6 and 14 months post-inoculation respectively; pSGTax_2531–18 contains tax sequences from leukemic PBMC isolated 18 months post-inoculation, and the pSGTax_2531-tum construct resulted from the insertion of lymphoma-derived tax sequences collected 18 months post-infection. pSGc is the empty control vector. Values represent the means of the results of triplicate samples. The results from a representative experiment of four independent experiments are shown.

Figure 2

Sheep S267: non-transformed blood-derived B-cells carry a potentially active provirus while virus and Tax expression are completely suppressed in the the co-existing malignant lymphoma B-cells. (A) Diagram of the BLV L267 provirus and major transcripts. The two LTRs and the gag, pro, pol, env, tax, and rex genes are represented. Vertical arrows indicate restriction sites in the L267 provirus: S, SacI; E, EcoRI. The position and direction of the PCR primers are indicated on the provirus map. The horizontal bar indicates the 8.4 kb-long region that was used as probe. Double lines represent the sequenced regions. The genomic, env, and tax/rex transcripts are represented below. Alternatively spliced RNAs are not shown. The translation products of the singly- and doubly-spliced transcripts and the positions of the RT-PCR primers are indicated. (B) Southern blot analysis following hybridization with a full-length BLV probe of SacI-digested DNA isolated from blood (BL267) and lymphoma (L267-1, -2 and -3) cells collected from S267 twenty nine months post-infection. SacI is indicative of the proviral load (upper row). Southern blot analysis of EcoRI-digested DNA indicates the presence of a single monoclonally-integrated provirus for all three lymphoma (L267) whereas the blood-derived BL267 cells display a polyclonal integration pattern (middle and lower panels). EcoRI-cleaved DNA generates two virus-host junction fragments for each integrated L267 provirus as illustrated in the diagram. Shown here in each lane are the fragments containing the 5' flanking genomic region. (C) Southern blot analysis of EcoRI-digested DNA isolated from the lymphoma (L267-1, -2, -3) and the cell lines derived from each of these lymphoma (CL267-1, -2, -3) cultured for four weeks. (D) RT-PCR analysis of RNA isolated from lymphoma-derived cell lines (CL267), 24 h-cultured blood-derived lymphocytes (BL267-24 h), fresh lymphoma (L267) and freshly isolated blood-derived lymphocytes (BL267). EnvA/Tax2 primers for the detection of the doubly-spliced tax/rex RNA were used. In the controls YR2 and YR2_LTaxSN, provirus is silent and active respectively. (E) PCR analysis using BLV tax-specific primer pair Tax1/Tax2 of DNA isolated from sheep inoculated with the various S267-isolated B-cell populations: six sheep were inoculated using either cultured (CL267) or fresh (L267) transformed B-cells, two sheep were injected with nontransformed PBMCs (BL267).