Interaction of EF-C/RFX-1 with the Inverted Repeat of Viral Enhancer Regions Is Required for Transactivation

Abstract The hepatitis B virus (HBV) and polyomavirus (Py) enhancer regions contain multiple cis-acting elements that contribute to enhancer activity. The EF-C binding site was previously shown to be an important functional component of each enhancer region. EF-C is a ubiquitous binding activity that interacts with an inverted repeat sequence in the HBV and Py enhancer regions. Although the EF-C binding site is required for optimal enhancer function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. With both the HBV and Py enhancer regions, EF-C stimulates the activity of adjacent enhancer elements in a synergistic manner. EF-C corresponds to RFX-1, a protein that binds to a conserved and functionally important site in major histocompatibility complex (MHC) class II antigen promoter regions. Interestingly, the RFX-1 binding site in MHC class II promoters only contains an EF-C half-site, maintaining one arm of the inverted repeat in an EF-C binding site. We have investigated the binding of purified EF-C and RFX-1 to sites in the Py and HBV enhancer regions that carry mutations that either disrupt one arm of the EF-C inverted repeat, or alter the spacing between the repeats. Our results show that the interaction of EF-C and RFX-1 with an intact inverted repeat is required for functional activity of these viral enhancer regions. Chemical footprinting and modification interference assays show that the interaction of EF-C and RFX-1 with the DRA MHC class II promoter truly represents half-site interaction, and that this binding is unstable. In contrast, the binding of EF-C and RFX-1 to the viral inverted repeats is stable. These results suggest that an additional activity may be required to stabilize EF-C/RFX-1 interaction with the MHC class II promoter, and that viral enhancer regions have evolved high affinity binding sites to sequester dimeric EF-C/RFX-1.