Genetic variability and in vitro transcriptional permissibility of primary ovine beta-retrovirus promoter isolates

Abstract

To assess genomic sequence conservation and variation in the proviral promoter of enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) in tissue samples from 3 sheep with nasal adenocarcinoma associated with ENTV and 3 sheep with pulmonary adenocarcinoma associated with JSRV and to identify a cell culture system that supports transcriptional activity of the ENTV and JSRV viral promoters. 6 adult sheep. Standard PCR procedures for detection of the ENTV and JSRV long terminal repeat (LTR) promoter region were performed on samples from the 3 nasal adenocarcinomas and 3 pulmonary adenocarcinomas, respectively. The LTRs were cloned into shuttle vectors, amplified, sequenced, and analyzed. The cloned LTR regions were transferred into reporter plasmids and multiple human and ruminant cell lines, and primary cells were transfected with the promoter-reporter plasmids. The viral promoter activity was evaluated by use of an in vitro β-galactosidase reporter assay. Each isolate had a unique nucleotide sequence. Single nucleotide polymorphisms were the most common LTR mutation and rarely occurred at transcription factor binding sites. Relative to ENTV, the JSRV promoter isolates had a conserved 66-bp U3 insertion, including the lung-specific transcription factor HNF-3β binding site. Among the cell lines used, human embryonic kidney (293T) and goat synovial membrane cells supported promoter transcription. The LTRs of ENTV and JSRV have extensive blocks of sequence conservation. Human 293T and goat synovial membrane cell lines may be suitable in vitro cell culture systems for further research of viral promoter functions.