Synergistic inhibition of human cytomegalovirus replication by interferon-alpha/beta and interferon-gamma

Abstract

Background: Recent studies have shown that gamma interferon (IFN-gamma) synergizes with the innate IFNs (IFN-alpha and IFN-beta) to inhibit herpes simplex virus type 1 (HSV-1) replication in vitro. To determine whether this phenomenon is shared by other herpesviruses, we investigated the effects of IFNs on human cytomegalovirus (HCMV) replication.

Results: We have found that as with HSV-1, IFN-gamma synergizes with the innate IFNs (IFN-alpha/beta) to potently inhibit HCMV replication in vitro. While pre-treatment of human foreskin fibroblasts (HFFs) with IFN-alpha, IFN-beta or IFN-gamma alone inhibited HCMV plaque formation by approximately 30 to 40-fold, treatment with IFN-alpha and IFN-gamma or IFN-beta and IFN-gamma inhibited HCMV plaque formation by 163- and 662-fold, respectively. The generation of isobole plots verified that the observed inhibition of HCMV plaque formation and replication in HFFs by IFN-alpha/beta and IFN-gamma was a synergistic interaction. Additionally, real-time PCR analyses of the HCMV immediate early (IE) genes (IE1 and IE2) revealed that IE mRNA expression was profoundly decreased in cells stimulated with IFN-alpha/beta and IFN-gamma (approximately 5-11-fold) as compared to vehicle-treated cells. Furthermore, decreased IE mRNA expression was accompanied by a decrease in IE protein expression, as demonstrated by western blotting and immunofluorescence.

Conclusion: These findings suggest that IFN-alpha/beta and IFN-gamma synergistically inhibit HCMV replication through a mechanism that may involve the regulation of IE gene expression. We hypothesize that IFN-gamma produced by activated cells of the adaptive immune response may potentially synergize with endogenous type I IFNs to inhibit HCMV dissemination in vivo.

Figure 1

IFN-α, IFN-β and/or IFN-γ inhibit HCMV plaque formation on HFFs. HFFs were pre-treated with (A) vehicle or 100 IU/ml each of (B) IFN-α, (C) IFN-β, (D) IFN-γ, (E) IFN-α and IFN-γ or (F) IFN-β and IFN-γ. Monolayers were subsequently infected with 1000 PFU of HCMV strain Towne-GFP, and plaque numbers were determined 11 d p.i. by fluorescence microscopy. Plaques were determined by counting a minimum of 10 GFP-positive cells in one foci.

Figure 2

Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ) synergistically inhibit HCMV plaque formation on HFFs. (A) Viral plaque reduction assay. HFFs were treated with vehicle or increasing amounts of IFN-α (■), IFN-β (●), IFN-γ (▲), IFN-α and IFN-γ (□) or IFN-β and IFN-γ (○) prior to infection with 400 PFU of Towne-GFP (n = 3). Fold-inhibition in IFN-treated groups as compared to vehicle-treated groups is plotted as a function of IFN concentration (IU/ml). Significant differences in fold-inhibition for HFFs treated with combination IFNs relative to cells treated with individual IFNs are denoted by a single asterisk (P < 0.001, one-way ANOVA and Tukey's post hoc t test). (B) Illustration of a representative isobologram for a combination of two drugs. The solid line is the line of additivity. When the isobole lies below the line of additivity, the combinatorial effect of drug A and drug B is synergistic. When the isobole lies above the line of additivity, the combinatorial effect of drug A and drug B is antagonistic. Combination effect of (C) IFN-α and IFN-γ and (D) IFN-β and IFN-γ on HCMV plaque formation on HFFs was plotted in an isobologram. Values used to generate the concave isoboles were derived from a dose response curve and represent a combination dose required to elicit 95% (IC₉₅) inhibition of viral plaque formation on HFFs. The dashed line represents the theoretical line of additivity.

Figure 3

IFN-α, IFN-β and/or IFN-γ inhibit HCMV replication in HFFs. HFFs were treated with vehicle or 100 IU/ml of IFNs 12 h prior to infection with HCMV at a MOI of 2.5: (◆) vehicle, (■) IFN-α, (●) IFN-β, (▲) IFN-γ, (□) IFN-α and IFN-γ, (○) IFN-β and IFN-γ or (◇) GCV (100 μM). On the indicated d p.i., average viral titers (n = 3) were determined by a microtiter plaque assay. HFFs were inoculated for 2 h with serially diluted lysed cultures. Plaque numbers were determined 11 d p.i. by fluorescence microscopy. At 3 d p.i., all IFN treatments significantly reduced viral titers as compared to vehicle-treated cultures (P < 0.001, one-way ANOVA and Tukey's post hoc t test). At 4 d p.i., only cells treated with GCV or combination IFN treatments inhibited viral titers as compared to vehicle-treated HFFs (P < 0.001, one-way ANOVA and Tukey's post hoc t test). Significant reduction denoted by a single asterisk. Inset: Represents HCMV titers determined over 11 d for (◆) vehicle-treated and (○) IFN-β and IFN-γ-treated HFFs. The dashed line represents the lower limit of detection of the plaque assay (20 PFU/ml) used to measure viral titers.

Figure 4

Inhibition of HCMV by IFN-α, IFN-β and/or IFN-γ is not a result of decreased viral entry into cells. Ethidium bromide-stained IE exon 4 PCR products amplified from HCMV-infected HFFs pre-treated with either vehicle (A) or 100 IU/ml of IFN-α (B), IFN-β (C), IFN-γ (D), IFN-α and IFN-γ (E) or IFN-β and IFN-γ (F). From left to right, PCR products were amplified from H₂O control, 100 ng of uninfected (UI) HFF DNA or 100 ng of HCMV-infected HFF DNA harvested from cells inoculated for 2 h at MOIs of 0.3 to 30. GAPDH PCR products were run along side IE exon 4 PCR products and served as internal loading controls (data not shown).

Figure 5

IFN-α, IFN-β and/or IFN-γ inhibit HCMV IE mRNA expression. SYBR green real-time PCR analyses of IE1 and IE2 mRNA expression in vehicle- or IFN-treated HFFs 6 h p.i. (n = 3). Presented are fold-inhibition ± standard deviation in IE1 (■) and IE2 (□) mRNA expression in each treatment group. Differences in gene expression were determined as described in Methods.

Figure 6

IFN-α, IFN-β and/or IFN-γ inhibit HCMV IE protein expression. (A) HFFs were pre-treated with either vehicle (1) or 100 IU/ml of IFN-α (2), IFN-β (3), IFN-γ (4), IFN-α and IFN-γ (5) or IFN-β and IFN-γ (6) 12 h prior to infection with HCMV. At 12 h p.i., cells were harvested and equal amounts of total protein were examined for IE protein (IE72, IE86) expression by western blot analyses. (B-G) Vehicle- or IFN-treated cells were infected with HCMV and the nuclear proteins IE72/86 were detected by indirect immunofluorescence 5 d p.i. Representative images (100X) from cultures treated with (B) vehicle, (C) IFN-α, (D) IFN-β, (E) IFN-γ, (F) IFN-α and IFN-γ or (G) IFN-β and IFN-γ. Immunofluorescent labeling: HCMV IE72/86 – Alexa Fluor 568 (red), nucleus – DAPI (blue), overlaid (pink).