In vivo infection of sheep by bovine leukemia virus mutants

Abstract

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp3O glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.

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

“…Cell lines and cell culture L267 and YR2 BLV infected cell lines were kindly given by Anne Van den Broeke (GIGA-R, University of Liège, Belgium). 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).…”

Investigations of the Japanese Bovine Tumour Virus (BLV)-its Ability to Express Structural and Regulatory BLV Proteins

“…Mutations may be involved in the molecular events leading to BLV-induced (WILLEMS et al, 1993). In recent investigations genetic variations of LTR and env gene during pathogenesis in sheep were analysed (WILLEMS et al, 1995).…”

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

“…It should also identify insertion sites when only a small fraction of the cells carries the provirus. To test these potential limitations, we applied PCIP-seq to four samples from BLV infected sheep (experimental infection 19 ) and three cattle (natural infection) to explore its performance on polyclonal and low proviral load (PVL) samples and compared PCIP-seq to our previously published Illumina method 4 . PCIPseq revealed all samples to be highly polyclonal ( Supplementary Fig.…”

“…Briefly, the sheep were infected with the molecular clone pBLV344 19 test was used to determine adequate sample size and the study did not use blinding.…”