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. 2004 Mar;78(6):3190-5.
doi: 10.1128/jvi.78.6.3190-3195.2004.

Human cytomegalovirus stimulates cellular IKK2 activity and requires the enzyme for productive replication

Patrizia Caposio  1 , Michel Dreano, Gianni Garotta, Giorgio Gribaudo, Santo Landolfo
Affiliations

Human cytomegalovirus stimulates cellular IKK2 activity and requires the enzyme for productive replication

Patrizia Caposio et al. J Virol. 2004 Mar.

Abstract

Human cytomegalovirus (HCMV) exploits the host transcription factor NF-kappaB to enhance its own replication, dissemination, and reactivation from latency. Here we report that HCMV infection activates the upstream IkappaB kinase (IKK) complex and that its catalytic IKK2 subunit is required for HCMV-induced NF-kappaB activation, as well as the replication of different HCMV strains. These results indicate that IKK2 is essential for HCMV replication and emphasize the feasibility of blocking NF-kappaB activation as a way of inhibiting infection.

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Figures

FIG. 1.
FIG. 1.
HCMV infection activates cellular IKK. (A) HCMV infection rapidly increases IKK activity. HELF cells were growth arrested in low-serum medium for 48 h and then infected with HCMV AD169 (MOI of 10 PFU/cell) or mock infected. Whole-cell protein extracts were then prepared at the indicated times and assayed for IKK activity using GST-IκBα as the substrate (17). Endogenous IKK recovery after immunoprecipitation was determined by immunoblotting for IKK1 (IB: IKK1). The autoradiograms of two independent experiments are shown as representative (upper panel). Following autoradiography, band intensity was determined with a densitometer, and its quantitation is shown. The data are the means of four experiments ± the standard errors (error bars) (middle panel). EMSA analysis was performed with nuclear extracts prepared from HELF cells infected with HCMV AD169 (MOI of 5 PFU/cell) or mock infected (lower panel). (B) IKK2 is functionally required for HCMV-induced NF-κB-dependent gene expression. HELF cells were transiently cotransfected with 2 μg of the 5xNF-κB LUC indicator plasmid and increasing amounts of the expression vector for the dnIKK2 protein. After 18 h they were washed and then maintained in low-serum medium for 48 h. Thereafter, transfectants were infected with HCMV AD169 (MOI of 5 PFU/cell) or mock infected. Total cytoplasmic extracts were isolated at 18 h p.i. and assayed for luciferase activity. Reporter gene activity was normalized to the amount of plasmid DNA introduced into recipient cells by DNA dot blot analysis (1). Luciferase activity is expressed as induction relative to the basal level in cells transfected with the 5xNF-κB LUC, which was set at 1. The data are the means of three experiments ± the standard errors (error bars). The extracts from transfected cells were also subjected to immunoblotting analysis with the anti-IKK2, the anti-FLAG, or the anti-actin MAb. Extracts were from cells cotransfected with the p5xNB-κB and the empty pcDNA3 vector and then mock infected (lane1), cells cotransfected with p5xNB-κB and the empty vector and then infected with HCMV (lane 2), or cells cotransfected with p5xNB-κB and increasing concentrations (2 μg [lane 3], 4 μg [lane 4], or 6 μg [lane 5]) of the dnIKK2 vector and then infected with HCMV.
FIG. 2.
FIG. 2.
Inhibition of HCMV-induced NF-κB activation by the IKK2 inhibitor AS602868. (A) Inhibition of HCMV-induced IKK activation by AS602868. Quiescent HELF cells were infected with HCMV AD169 (MOI of 10 PFU/cell) or mock infected. Whole-cell protein extracts were then prepared at 1 h p.i. and assayed for IKK activity. Where indicated, immunopurified IKK complex was incubated with increasing concentrations of AS602868 during the assay (lanes 3 to 5). Extracts analyzed for IKK activity in lane 8 were from cells treated with 20 μM AS602868 1 h prior to and during infection. Endogenous IKK recovery after immunoprecipitation was determined by immunoblotting for IKK1 (IB: IKK1). The autoradiogram of a representative experiment is shown. (B) AS602868 inhibits the induction of NF-κB DNA binding activity following HCMV infection. Quiescent HELF cells were infected with HCMV AD169 (MOI of 5 PFU/cell) or mock infected. Nuclear extracts were then prepared at the indicated times and assayed for NF-κB activation by EMSA. Where indicated, cells were treated with 20 μM AS602868 1 h prior to and during infection. This experiment was repeated three times, and a representative autoradiogram is shown. (C) Inhibition of IKK2 activity inhibits HCMV-induced NF-κB-dependent transactivation. HELF cells were transiently transfected with 2 μg of p5xNF-κB LUC and 10 μg of carrier DNA (pBluescript SK). After 18 h, cells were washed and growth arrested in low-serum medium for 48 h. Thereafter, transfectants were infected with HCMV AD169 (MOI of 5 PFU/cell) or mock infected. Where indicated, cells were treated with different concentrations of AS602868 1 h prior to and during infection. Luciferase activity was measured at 18 h p.i. and is expressed as induction (n-fold) relative to the basal level measured in cells transfected with p5xNF-κB LUC and then mock infected, which was set at 1. The data shown are the means of three experiments ± the standard errors (error bars).
FIG. 3.
FIG. 3.
Effects of AS602868 on HCMV AD169 protein and DNA synthesis. (A) Expression of HCMV proteins is decreased by IKK2 inhibition. Growth-arrested HELF cells were infected with HCMV AD169 (MOI of 5 PFU/cell) or mock infected. Where indicated, they were treated with 20 μM AS602868 1 h prior to and during infection. Total cell extracts were prepared at the indicated times after infection, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (50 μg of protein per lane), and analyzed by immunoblotting with the anti-IEA, anti-UL44, or anti-UL99 MAbs. Actin immunodetection was performed as an internal control. (B) Effect of AS602868 on viral DNA replication. Quiescent HELF cells were infected with HCMV AD169 (MOI of 1 PFU/cell) or mock infected. Where indicated, cells were treated with increasing concentrations of AS602868 1 h prior to and during infection. At 96 h p.i., total genomic DNA was purified and twofold dilutions were immobilized on a hybridization membrane. The same filter was sequentially hybridized with 32P-labeled HCMV IE1 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probes. Hybridization signals were quantitated with a densitometer. (C) Reversibility of AS602868 treatment. Quiescent HELF cells were infected with HCMV AD169 (MOI of 1 PFU/cell) or mock infected. Where indicated, cells were treated with 10 μM AS602868 1 h prior to and during infection. At 96 h p.i., where indicated, AS602868 was removed from the culture medium and the infection was allowed to continue for another 24 or 48 h. At 120 or 144 h p.i., total genomic DNA was purified and analyzed by dot blot hybridization.
FIG. 4.
FIG. 4.
Antiviral effects of the IKK2 inhibitor AS602868. (A) Inhibitory effect of AS602868 on HCMV AD169 replication in HELF cells. Growth-arrested HELF cells were infected with HCMV AD169 (MOI of 1 PFU/cell) or mock infected. Where indicated, cells were treated with increasing concentrations of AS602868 1 h prior to and during infection until an extensive viral cytopathic effect was observed in the untreated control. Supernatants of cell suspension were then assayed for infectivity by standard plaque reduction assay on HELF cells. The number of plaques was plotted as a function of drug concentration, and the IC50 and IC90 were determined. Values are the means of two independent determinations. To determine cell viability, HELF cells were growth arrested in low-serum medium and then exposed to increasing concentrations of AS602868. After four days, the number of viable cells was determined by the MTT method, as previously described (21). (B) Inhibitory effect of AS602868 on GCV-resistant HCMV VR5438 and VR6264 strains. Quiescent HELF cells were infected with HCMV VR5438 or HCMV VR6264 (MOI of 1 PFU/cell) or mock infected. Where indicated, cells were treated with increasing concentrations of AS602868 1 h prior to and during infection until an extensive viral cytopathic effect was observed in the untreated control. The extent of VR5438 or VR6264 replication was then assessed by titrating the infectivity of supernatants of cell suspensions by the IE antigen indirect immunoperoxidase staining technique (8). The number of plaques was plotted as a function of drug concentration, and the IC50 and IC90 values were determined.
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