The 6-Aminoquinolone WC5 Inhibits Human Cytomegalovirus Replication at an Early Stage by Interfering with the Transactivating Activity of Viral Immediate-Early 2 Protein

Abstract

WC5 is a 6-aminoquinolone that potently inhibits the replication of human cytomegalovirus (HCMV) but has no activity, or significantly less activity, against other herpesviruses. Here we investigated the nature of its specific anti-HCMV activity. Structure-activity relationship studies on a small series of analogues showed that WC5 possesses the most suitable pattern of substitutions around the quinolone scaffold to give potent and selective anti-HCMV activity. Studies performed to identify the possible target of WC5 indicated that it prevents viral DNA synthesis but does not significantly affect DNA polymerase activity. In yield reduction experiments with different multiplicities of infection, the anti-HCMV activity of WC5 appeared to be highly dependent on the viral inoculum, suggesting that WC5 may act at an initial stage of virus replication. Consistently, time-of-addition and time-of-removal studies demonstrated that WC5 affects a phase of the HCMV replicative cycle that precedes viral DNA synthesis. Experiments to monitor the effects of the compound on virus attachment and entry showed that it does not inhibit either process. Evaluation of viral mRNA and protein expression revealed that WC5 targets an event of the HCMV replicative cycle that follows the transcription and translation of immediate-early genes and precedes those of early and late genes. In cell-based assays to test the effects of WC5 on the transactivating activity of the HCMV immediate-early 2 (IE2) protein, WC5 markedly interfered with IE2-mediated transactivation of viral early promoters. Finally, WC5 combined with ganciclovir in checkerboard experiments exhibited highly synergistic activity. These findings suggest that WC5 deserves further investigation as a candidate anti-HCMV drug with a novel mechanism of action.

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

“…We previously observed that WC5 is able to inhibit the IE2-dependent transactivation of two E genes of HCMV, i.e., UL112-113 and UL54, both in transfected cells and in the context of viral infection (31). Thus, to gain further insights into the mechanism of this inhibitory activity, we wished to identify the minimal portion of an E gene promoter activated by IE2 still sensitive to WC5 inhibition.…”

“…We previously observed that WC5 interferes with the transactivating activity of IE2 on two HCMV E gene promoters (31). Since IE2 regulates gene expression by both protein-protein interactions and direct binding to specific DNA sequences within E-responsive promoters (10), we first wished to determine whether the inhibitory effect of WC5 might be due to interference with two protein-protein interactions that are required for the transactivating activity of IE2, such as the interaction with itself and with cellular TBP.…”

“…In this regard, we have identified a 6-aminoquinolone (6-AQ) derivative termed WC5, endowed with a specific anti-HCMV activity (30,31). WC5 and some analogues have been demonstrated to inhibit the IE2-induced transcriptional activation of two HCMV E gene promoters, i.e., UL112-113 and UL54, in a specific cell-based assay (31)(32)(33).…”

Approaches for the Generation of New Anti-cytomegalovirus Agents: Identification of Protein–Protein Interaction Inhibitors and Compounds Against the HCMV IE2 Protein

“…The EGFP-expressing cell lines show a specific and inducible dose-and time-dependent EGFP response to either HCMV infection or constitutive IE2 expression that can be visually assessed by fluorescence microscopy or evaluated by automated fluorometry (8). The reliability of these reporter cell lines for assessing virus-inhibitory effects in concentration-and time-dependent fashions after HCMV infection or IE2 transfection has been demonstrated by treatment with fomivirsen, an antisense oligodeoxynucleotide designed to block IE2 expression (8,9), and with WC5, a novel 6-aminoquinolone endowed with potent inhibitory activity for HCMV replication (10). Thus, these EGFP-based cell lines can be exploited as a tool to screen antiviral compounds that specifically target IE2 expression and/or function.…”

Sulfated Derivatives of Escherichia coli K5 Capsular Polysaccharide Are Potent Inhibitors of Human Cytomegalovirus

“…The effects of K5 derivatives on HCMV attachment were analyzed as previously described (20). Briefly, 100 PFU of HCMV AD169 and various concentrations of K5 derivatives or heparin as a control were added to HFF monolayers prechilled at 4°C and incubated for either 2 or 4 h at 4°C.…”