Transcriptional regulation of TT virus: promoter and enhancer regions in the 1.2-kb noncoding region.

Since the discovery of TT virus (TTV) in 1997, its mechanism of transcriptional control has remained unsolved. Molecular analysis points at the 1.2-kb noncoding region (NCR) as being responsible for transcriptional control. The 5' terminus of TTV mRNA was located at nt 114 using the primer extension method (nt 114 will be referred to as position +1). This employed the PE1 primer, designed to start approximately 100 nt downstream of the predicted initiation site. Overall promoter and enhancer activity of the NCR was analyzed using dual luciferase assays in K562, Jurkat, U937, A549, HepG2, Huh7, and HeLaS3 cells. Of those tested, K562 showed the highest relative luciferase activity of 31.1, and activity in HepG2 (14.6) was significantly higher than that in Huh7 (2.8). Fragments of <250 nt length, spanning the NCR, were inserted into a luciferase vector possessing an SV40 promoter. Fragments F5(-542/-311) and F6(-310/-197) showed promoter-enhancing activities of >6.0 by insertion not only in the sense orientation, but also both in the antisense orientation and downstream of the luciferase gene. The 5' deletion of NCR from -1201 to -370 resulted in no significant decrease in the level of luciferase activity. A gradual decrease in the activity of the 5'-deletion mutants from position -370 through -155 was consistent with the loss of enhancer binding sites detected during fragment analysis. A further deletion at position -76 completely abolished luciferase expression, indicating that region -154/-76 contains the critical regulatory element for functioning of the TTV promoter.