In Vivo Rescue of a Silent tax -Deficient Bovine Leukemia Virus from a Tumor-Derived Ovine B-Cell Line by Recombination with a Retrovirally Transduced Wild-Type tax Gene

Abstract

The lack of bovine leukemia virus (BLV) expression is a consistent finding in freshly isolated ovine tumor cells and in the B-cell lines derived from these tumors. In order to gain further insight into the mechanisms of BLV silencing in these tumors, we have used the YR2 B-cell line, which was derived from the leukemic cells of a BLV-infected sheep. This cell line contains a single, monoclonally integrated, silent provirus, which cannot be reactivated either by stimulation in vitro or by in vivo injection of the tumor cells or cloned proviral DNA in sheep. Sequence analysis of the tax gene from the YR2 cell line identified two G-to-A transitions (G 7924 to A 7924 and G 8149 to A 8149 ) that result in E-to-K amino acid changes at positions 228 and 303 in the Tax protein.Following retroviral vector-mediated transfer of a wild-type tax gene into YR2 cells, we showed that BLV mRNA, viral proteins, and virions were produced, demonstrating that the cellular factors required for virus expression were present in the original YR2 cell line. Injection of this transduced YR2 cell line in sheep led to the rescue of replication-competent BLV proviruses. The integrated competent proviruses exhibited unique chimeric tax genes, which arose from homologous recombination between the transduced wild-type tax and the YR2-derived tax sequences. Furthermore, in one of these functional recombinant proviruses, only the A 8149 -to-G 8149 reversion was present, providing clear evidence that the defect underlying the silent phenotype in YR2 cells results from a single C-terminal E 303 -to-K 303 amino acid substitution in the BLV Tax protein. Our observations suggest that a single strategically located mutation in tax provides a mechanism for BLV inactivation in B-cell tumors.

Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome

“…L267 is a clonal lymphoma-derived B-cell line established from a BLV-infected sheep (S267) injected with naked proviral DNA of an infectious BLV variant (30), whose provirus displays a wild-type sequence (23,31). The L267 LTaxSN cell line results from the transduction of native L267 with the pLTaxSN retroviral vector expressing the tax cDNA following cocultivation with the PG13 LTaxSN producer cell line and G418 selection of transduced cells (31,32). YR2 is a cloned Blymphoid cell line established from peripheral blood lymphocytes (PBLs) isolated from a BLVinfected sheep (33), containing a single monoclonally integrated mutated silent provirus (5,32).…”

“…The L267 LTaxSN cell line results from the transduction of native L267 with the pLTaxSN retroviral vector expressing the tax cDNA following cocultivation with the PG13 LTaxSN producer cell line and G418 selection of transduced cells (31,32). YR2 is a cloned Blymphoid cell line established from peripheral blood lymphocytes (PBLs) isolated from a BLVinfected sheep (33), containing a single monoclonally integrated mutated silent provirus (5,32). These cell lines were maintained in Opti-MEM medium (Life Technologies) supplemented with 10% FBS, 1mM sodium pyruvate, 2 mM glutamine, non-essential amino acids and 100µg/ml kanamycin.…”

Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the associated provirus in thousands of cells with long reads

“…Firstly, the presence of the recurrent mutations at the first base of codon 303 in the viral protein Tax, a central player in the biology of both HTLV-1 32 and BLV 34 . It has previously been reported that this mutation causes an E-to-K amino acid substitution which ablates the transactivator activity of the Tax protein 21 . Collectively, these observations suggest this mutation confers an advantage to clones carrying it, possibly contributing to immune evasion, while retaining Tax protein functions that contribute to clonal expansion.…”

“…The majority of the variants observed were G-to-A transitions (results in E-to-K amino acid change), however we also observed G-to-T (E-to-STOP) and G-to-C (E-to-Q) transversions. It has been previously shown that the G-to-A mutation abolishes the Tax proteins transactivator activity 21,22 . The repeated selection of variants in this specific position suggests that they reduce viral protein recognition by the immune system, while preserving the Tax proteins other proliferative properties.…”

“…In this instance, we would expect to see the same position mutated in multiple individuals. One potential example is found in the first base of codon 303 (position 8155) of the viral protein Tax, a potent viral transactivator, stimulator of cellular proliferation and highly immunogenic 21 . A variant was observed at this position in multiple proviruses (14, 18 and 2 respectively) in the ovine samples 233, 221 (022016) & 220, in one provirus from bovine 1053 and in two for bovine 1439 (Fig.…”