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Journal of Virology, February 1999, p. 1054-1065, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Anne Van Den Broeke,1,2,* Claude Bagnis,3 Malgorzata Ciesiolka,1,2 Yvette Cleuter,2 Hans Gelderblom,4 Pierre Kerkhofs,5 Philip Griebel,6 Patrice Mannoni,3 and Arsene Burny1,2

Laboratoire d'Investigation Clinique et d'Oncologie Expérimentale, Institut Bordet, Université Libre de Bruxelles, 1000 Brussels,1 Département de Biologie Moléculaire, Université Libre de Bruxelles, 1640 Rhode St-Genèse,2 and Département de Virologie Bovine, Centre d'Etude et de Recherches Vétérinaires et Agrochimiques, 1180 Uccle,5 Belgium; Laboratoire de Thérapie Génique, Institut Paoli-Calmettes, 13009 Marseille, France3; Robert Koch-Institut, 13353 Berlin, Germany4; and Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan, Canada6

Received 26 May 1998/Accepted 20 October 1998

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 (G7924 to A7924 and G8149 to A8149) 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 A8149-to-G8149 reversion was present, providing clear evidence that the defect underlying the silent phenotype in YR2 cells results from a single C-terminal E303-to-K303 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.

* Corresponding author. Mailing address: Université Libre de Bruxelles, Laboratoire de Chimie Biologique, rue des Chevaux, 67, B-1640 Rhode St-Genèse, Belgium. Phone: 32 2 6509826. Fax: 32 2 6509839. E-mail: avdb{at}dbm.ulb.ac.be.

Journal of Virology, February 1999, p. 1054-1065, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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