Discordance between bovine leukemia virus tax immortalization in vitro and oncogenicity in vivo

Abstract

Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

FIG. 1

Evolution of proviral loads in sheep infected with the BLV Tax mutants. Three sheep (11, 292, and 293) were injected with plasmid pBLVIX, which contains an infectious and pathogenic BLV provirus (clone 344). Four other animals (103, 104, 296, and 480) were infected with pBLVTax106+293, which is isogenic to pBLVIX except for two serine-to-alanine mutations in the tax gene. Blood was extracted by jugular venipuncture at regular times after seroconversion (4, 6, 15, and 30 months), and partially purified DNA was prepared from the corresponding lysates. A fraction corresponding to 5 μl of blood was amplified by 22 cycles of PCR using two primers flanking the tax gene, and the resulting DNAs were analyzed by Southern blotting using a tax probe. Under these conditions, the PCRs were semiquantitative, as shown by 10-fold dilutions (1×, 10×, 100×, and 1,000×) of lysate 480 at 15 months. In some lanes (∗), the DNAs had to be isolated from smaller volumes of blood (50 μl instead of 500) because of the very high lymphocyte counts. Sheep 113 is an uninfected (NI) animal used as a negative control for PCR contaminations. Three samples are lacking at 30 months (†) because sheep 11, 103, and 480 died at about 19 to 20 months after seroconversion. Sheep 103 died because of enterotoxemia, whereas the other animals succumbed with leukemia or lymphosarcoma.

FIG. 2

Evolution of lymphocyte counts in BLV-infected sheep. Sheep were infected with the pBLVTax106+293 recombinant (TAX) (animals 103, 104, 296, and 480) or with viruses exhibiting wild-type (WT) behavior during pathogenesis (plasmid pBLVIX in sheep 8, 11, 247, 292, and 293; pBLV344 in 235; and pBLVgag150 in 175). Blood samples were extracted at regular intervals (routinely every month), and the number of leukocytes per microliter was determined by using a Coulter counter ZN. The lymphocyte counts (in parentheses) were deduced from these numbers after microscopic determination of the blood formula.

FIG. 3

(A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by Percoll gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.

FIG. 4

Direct sequencing of codons surrounding alanines 106 and 293 of the tax gene in four sheep infected by the BLV Tax mutant. Blood was extracted by jugular venipuncture of sheep 103, 104, 296, and 480 infected with provirus pBLVTax106+293. After lysis, partially purified DNA was amplified by PCR using two primers flanking the tax gene. The resulting amplicons were then subjected to direct sequencing by PCR and migrated onto a denaturing polyacrylamide gel. The sequences surrounding alanines 106 and 293 are indicated.