Characterization of enhancer elements and their mutations in the long terminal repeat of feline endogenous RD-114 proviruses.

To locate the enhancer regions of the feline endogenous RD-114 long terminal repeat (LTR), we examined expression of the chloramphenicol acetyltransferase gene driven by various segments of the U3 region from two different proviral loci (CRL3 and CR1). Transient expression assays demonstrated that the primary signal sequence for transcription enhancement was located within the 63-base-pair (bp) element of the CRL3 DNA occurring between positions -184 and -121 from the CAP site (+1), whereas the similar region of CR1 was almost inactive. This element from both CRL3 and CR1 contained a single 30-bp sequence (direct repeat [DR]-B2) found in duplicate tandem copies in the LTR of the infectious RD-114 provirus. Two 9-bp inverted repeats marked the DR-B unit of the active element, and a prominent base deletion in one of these repeats in CR1 DNA appeared to be related to loss of enhancer activity. Another segment of CRL3 (-296 to -184), also displaying enhancer function, contained tandem repeated sequences (DR-A1 and DR-A2). The Dr-A2 unit, which lacked the 5' 20-bp sequence of the 47-pb DR-A1, could not function as an enhancer by itself, but it contributed to enhancer effects in cooperation with either the DR-A1 or DR-B2 region. The CR1 LTR contained a single DR-A1 sequence with extensive mutations, and the region (-313 to -181) containing this DR-A1 unit was nonfunctional, similar to the DR-B2 region of CR1. Site-directed mutagenesis analysis of another enhancer element, an octamer motif occurring between CAAT and TATA boxes of all RD-114 LTRs sequenced, revealed that this element was necessary for full enhancer function of the U3 region but with a variable effect, depending on the cell types in which chloramphenicol acetyltransferase expression was determined.