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.

FIG. 1.

WC5 does not significantly inhibit the activity of HCMV DNA polymerase in vitro. The effects of increasing concentrations of WC5 (filled symbols), and of FOS as a control (open symbols), on the activity of the purified, baculovirus-expressed HCMV DNA polymerase catalytic subunit UL54 in the absence (circles) and in the presence (squares) of the accessory subunit UL44 were determined by filter-based DNA polymerase assays. The data shown are means ± standard deviations (error bars) for four independent experiments.

FIG. 2.

WC5 prevents viral DNA synthesis in HCMV-infected cells. HFF were either infected with HCMV AD169 (MOI, 0.1) or mock infected (Mock) and were then treated with either WC5, GCV, or DMSO as a control. At 72 h p.i., total DNA was extracted, and qPCR was performed with appropriate IE2 and β-actin primers. HCMV genomic copies were normalized to the cellular β-actin gene copies. Data shown are means ± standard deviations (error bars) for three independent experiments performed in duplicate.

FIG. 3.

WC5 does not interfere with the adsorption and entry of HCMV. (A) Effects on attachment. HCMV AD169 was allowed to attach to HFF at 4°C in the presence of various concentrations of WC5 (▪) or heparin (•) as a control. (B) Effects on entry. HCMV AD169 was attached to cells at 4°C; unattached virus was removed; and the cells were incubated for 2 h at 37°C in the presence of various concentrations of WC5 (▪) or ODN CpG 2006 (•) as a control. The data shown in both panels are means ± standard deviations for three experiments carried out in duplicate.

FIG. 4.

Effects of addition and removal of WC5 at different time-points after HCMV infection. (A) Effects of time of addition of WC5 and GCV on HCMV yield. HFF were infected with HCMV AD169 at an MOI of 1 and were then treated with WC5 (▪) or GCV (• at 0, 1, 2, 6, 9, 12, 24, 32, 48, 56, 60, 72, 78, or 84 h p.i. The antiviral activity of each compound is expressed as a percentage of the virus titer in the untreated infected samples. (B) Effects of time of removal of WC5 or GCV on HCMV yield. HFF were infected with HCMV AD169 at an MOI of 1 and were treated with WC5 (▪) or GCV (•). At 1, 2, 6, 9, 12, 24, 32, 36, 48, 58, 72, 78, 84, or 96 h p.i., the cells were rinsed to remove the compound, and fresh medium was added; infected cultures with no compound were treated identically to serve as controls. In both panels, the data shown are means ± standard deviations for three experiments carried out in duplicate.

FIG. 5.

WC5 inhibits transcription from viral E and L genes but not from viral IE genes. HFF were either infected with HCMV AD169 (MOI, 0.1) or mock infected and were treated with WC5 or DMSO as a control. Infected cell samples treated with ActD, CHX, and FOS were included as controls for inhibition of IE, E, and L gene expression, respectively. At 16 and 48 h p.i., total RNA was extracted and reverse transcribed. PCRs were then carried out with primers for the UL122 and UL123 (IE), UL54 and UL84 (E), and UL55 and UL75 (L) genes. Human β-actin was included as a control. PCR products were analyzed in ethidium bromide-stained agarose gels.

FIG. 6.

Effects of WC5 on the expression of HCMV IE, E, and L proteins. HFF were either infected with HCMV AD169 (MOI, 0.1) or mock infected and were then treated with WC5 or with DMSO as a control. At 24, 48, and 72 h p.i., whole-cell extracts were prepared, fractionated by SDS-PAGE, and analyzed by Western blotting (WB) with anti-IEA (IE1 and IE2), anti-UL44, and anti-UL55 antibodies. Controls for inhibition of gene expression were ActD (24 h), CHX (48 h), and FOS (72 h). Immunodetection of cellular β-actin was also included as an internal control.

FIG. 7.

WC5 inhibits the transactivation of E promoters by IE2. (A) U373-MG cells stably transfected with the pUL54-EGFP or the pUL112/113-EGFP plasmid were either infected with HCMV AD169 (MOI, 0.1) or mock infected. Cells were then treated with either WC5, fomivirsen, or DMSO as a control. At 48 h p.i., the cells were examined by confocal fluorescence microscopy to evaluate the number of EGFP-expressing cells. (B) 293T cells either were transiently transfected with the UL54-EGFP or the UL112/113-EGFP reporter plasmid alone or in combination with pSGIE86 or were mock transfected (Mock). Cells were then treated with WC5, fomivirsen, or DMSO as a control. At 24 h after transfection, the cells were analyzed for EGFP expression by confocal fluorescence microscopy. Representative images are shown for both panels.

FIG. 8.

Effects of combinations of WC5 and GCV on HCMV replication. HFF were infected with HCMV AD169 and were then treated with different concentrations of WC5 either alone or combined with different concentrations of GCV. The data for each compound are reported as the fractional inhibitory concentration (FIC), calculated by dividing the EC₅₀ of compound X in combination with compound Y by the EC₅₀ of compound X alone. The solid and broken lines represent the unit lines for FICs equal to 1 and 0.5, respectively. The FIC index, which is the FIC of compounds X and Y combined, is given for every combination of concentrations. A FIC index between 0.5 and 0.9 is suggestive of partial synergism, whereas a FIC index of <0.5 indicates significant synergism. The results are from a single experiment performed in quadruplicate, representative of two independent experiments.