Functional characterization of a piscine retroviral promoter

Walleye dermal sarcoma virus (WDSV) is a retrovirus aetiologically associat ed with a multifocal skin tumour of walleye. Tumours synchronously develop on 27% of fish and regress seasonally; their severity is influenced by wate r temperature. To functionally characterize the LTR of WDSV, the LTR was fu sed to the luciferase reporter gene. WDSV LTR was found to be transcription ally active in both fish and mammalian cells. WDSV LTR deletion mutants wer e constructed to identify specific regions that were functionally important in modulating viral gene expression and in temperature responsiveness. The 5' end 60 bp, which contain a putative ecdysone-response element also pres ent in another fish retrovirus, positively modulated transcription from the WDSV LTR at 25 degrees C, but not at lower temperatures. A 13 bp region (n t -288 to -275) comprising a putative activator protein-1 element was neces sary for maintaining WDSV LTR activity at all temperatures. In marked contr ast to the short direct repeats found in mammalian retroviral LTRs, five 5 bp direct repeats (nt -336 and -272) were found to negatively regulate tran scription from the WDSV LTR, A region spanning nt -440 to -218 stimulated t he activity of a heterologous mammalian promoter in an orientation-dependen t manner, modestly in fish cells (1.3- to 2-fold), but markedly (3.7- to 5. 1-fold) in mammalian cells. Our results strongly suggest that the putative promoter elements present in the WDSV LTR function differentially in a temp erature-specific manner and that complex interactions between these element s modulate WDSV LTR activity in response to temperature changes.