CHARACTERIZATION OF THE SEQUENCES OF THE HUMAN CYTOMEGALOVIRUS ENHANCER THAT MEDIATE DIFFERENTIAL REGULATION BY NATURAL AND SYNTHETIC RETINOIDS

Evidence exists to suggest that human cytomegalovirus (hCMV) may oppor tunistically use retinoic acid (RA) to advance its own replication, in which transcriptional activation of the viral major immediate-early p romoter is a crucial control point. We demonstrate that the enhancer o f the viral promoter contains three RA-response-elements that cooperat e in mediating RA activation, These elements are direct repeats of two sequence motifs separated by 2 bp (DR2 site, REa) and 5 bp (DR5 sites , REb and c). DNA-binding experiments revealed that each of these elem ents bind RA receptor (RAR)-retinoid X receptor (RXR) heterodimers mor e efficiently than either homodimer. Apparent equilibrium dissociation constants of RAR-RXR heterodimers for sites REa, REb, and REc were es timated to be 5 nM, 10 nM, and 20 nM, respectively. The level of contr ibution of each of these elements to RA inducibility correlated with t he strength of binding by RAR-RXR heterodimers to each site. These exp eriments demonstrate that RAR and RXR are necessary for RA responsiven ess of the viral promoter, Using synthetic RA analogs, which selective ly activate RARs and RXRs, the RAR partner within the heterodimeric co mplex appeared to be sufficient while the RXR partner was insufficient to independently activate transcription. However, joint activation of RARs and RXRs indicated that RXRs (in the presence of a transcription ally active RAR) could contribute to transactivation. This restricted co-dependent ligand activation of RXR varied depending on the particul ar response element and the cell context. These studies further indica te that signaling of retinoid receptors (in particular RAR) by RA play s an important role in modulating hCMV infection.