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

Abstract

Objective 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. Animals 6 adult sheep. Procedures 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. Results 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. Conclusions and Clinical Relevance 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.

Triterpenes from Poria cocos are revealed as potential retinoid X receptor selective agonists based on cell and in silico evidence

“…The 293T cell line is a model in vitro cell culture system for research of target gene promoter transcription because of its high efficiency in cell transfection (Eckstrand et al, ). Approximately 24 hr before transfection, the 293T cells were plated at a density of 40,000 cells/well.…”

16α-Hydroxytrametenolic Acid from Poria cocos Improves Intestinal Barrier Function Through the Glucocorticoid Receptor-Mediated PI3K/Akt/NF-κB Pathway

“…The determination of transcriptional activity was performed in both human colonic epithelial cell line Caco-2 and embryonic kidney cell line 293T; the latter is widely used in a transcriptional activation assay because of its high efficiency in transfection. 39 Compared to the control, the luciferase activities with treatment of HTA, DTA, DEA, PPAC, and PA in Caco-2 cells were increased by around 17-, 11-, 7-, 14-, and 3-fold, respectively., The luciferase activities with treatment of HTA, DTA, DEA, PPAC and PA in 293T cells were increased by nearly 14-, 13-, 11-, 8-, and 3fold, respectively (Figure S1). Hence, HTA-treated cells had the strongest luciferase activity in both Caco-2 and 293T cell lines.…”