The 6-Aminoquinolone WC5 Inhibits Different Functions of the Immediate-Early 2 (IE2) Protein of Human Cytomegalovirus that are Essential for Viral Replication

Human cytomegalovirus (HCMV) IE2 protein is a multifunctional factor essential for viral replication. IE2 modulates both viral and host gene expression, deregulates cell cycle progression, acts as an immunomodulator, and antagonizes cellular antiviral responses. Based on these facts, IE2 has been proposed as an important target for the development of innovative antiviral approaches. We previously identified the 6-aminoquinolone WC5 as a promising inhibitor of HCMV replication and here we report the dissection of its mechanism of action against viral IE2 protein. By GST-pulldown assays, mutagenesis, cell-based assays, and electrophoretic mobility shift assays, we demonstrated that WC5 did not interfere with IE2 dimerization, its interaction with TBP, and the expression of a set of cellular genes that are stimulated by IE2. On the contrary, WC5 targets the regulatory activity exerted by IE2 on different responsive viral promoters. Indeed, WC5 blocked the IE2-dependent negative regulation of the Major Immediate-Early promoter by preventing IE2 binding to the crs element. Moreover, WC5 reduced the IE2-dependent transactivation of a series of indicator constructs driven by different portions of the Early UL54 gene promoter and also inhibited the transactivation of the murine CMV early E1 promoter by the IE3 protein, the MCMV IE2 homolog. In conclusion, our results indicate that the overall anti-HCMV activity of WC5 depends on its ability to specifically interfere with IE2-dependent regulation of viral promoters. Importantly, our results suggest that this mechanism is conserved in the murine CMV, thus paving the way to further preclinical evaluation in an animal model.