title: The 6-Aminoquinolone WC5 inhibits different functions of the immediate-early 2 (IE2) protein of human cytomegalovirus that are essential for viral replication.
abstract: The human cytomegalovirus (HCMV) immediate-early 2 (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 the viral IE2 protein. Using glutathione S-transferase (GST) pulldown assays, mutagenesis, cell-based assays, and electrophoretic mobility shift assays, we demonstrated that WC5 does not interfere with IE2 dimerization, its interaction with TATA-binding protein (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 it also inhibited the transactivation of the murine CMV early E1 promoter by the IE3 protein, the murine cytomegalovirus (MCMV) IE2 homolog. In conclusion, our results indicate that the overall anti-HCMV activity of WC5 depends on its ability to specifically interfere with the IE2-dependent regulation of viral promoters. Importantly, our results suggest that this mechanism is conserved in murine CMV, thus paving the way for further preclinical evaluation in an animal model.
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Mercorelli
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Luganini
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Muratore
authors.affiliation: Department of Chemistry and Technology of Drugs, University of Perugia, Perugia, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Massari
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy.
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Terlizzi
authors.affiliation: Department of Chemistry and Technology of Drugs, University of Perugia, Perugia, Italy.
authors.forename: Oriana
authors.initials: O
authors.lastname: Tabarrini
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy giorgio.gribaudo@unito.it giorgio.palu@unipd.it arianna.loregian@unipd.it.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Gribaudo
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy giorgio.gribaudo@unito.it giorgio.palu@unipd.it arianna.loregian@unipd.it.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Palù
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy giorgio.gribaudo@unito.it giorgio.palu@unipd.it arianna.loregian@unipd.it.
authors.forename: Arianna
authors.initials: A
authors.lastname: Loregian
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology*; D000956:Antigens, Viral; D000998:Antiviral Agents / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints; D002460:Cell Line; D019927:Cyclin E / Q000235:genetics; D003587:Cytomegalovirus / Q000187:drug effects / Q000254:growth & development*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000037:antagonists & inhibitors*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D015363:Quinolones / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000634:Aminoquinolines; D000956:Antigens, Viral; D000998:Antiviral Agents; D019927:Cyclin E; D017874:Immediate-Early Proteins; D015363:Quinolones; C109367:UL54 protein, Human herpesvirus 5; D014764:Viral Proteins; C586457:WC5 compound; C044582:immediate-early proteins, cytomegalovirus; D004259:DNA-Directed DNA Polymerase