Global suppression of the host antiviral response by Ebola- and Marburgviruses: increased antagonism of the type I interferon response is associated with enhanced virulence

Abstract

We studied the effect of filovirus infection on host cell gene expression by characterizing the regulation of gene expression responses in human liver cells infected with Zaire Ebolavirus (ZEBOV), Reston Ebolavirus (REBOV), and Marburgvirus (MARV), using transcriptional profiling and bioinformatics. Expression microarray analysis demonstrated that filovirus infection resulted in the up-regulation of immune-related genes and the down-regulation of many coagulation and acute-phase proteins. These studies further revealed that a common feature of filovirus virulence is suppression of key cellular antiviral responses, including TLR-, interferon (IFN) regulatory factor 3-, and PKR-related pathways. We further showed that ZEBOV and MARV were more potent antagonists of the IFN response and inhibited the expression of most of the IFN-stimulated genes (ISGs) observed in mock-infected IFN-alpha-2b treated cells, compared to REBOV infection, which activated more than 20% of these ISGs. Finally, we examined IFN-related gene expression in filovirus-infected cells treated with IFN-alpha-2b. These experiments revealed that a majority of genes induced in mock-infected cells treated with type I IFN were antagonized in treated ZEBOV- and MARV-infected cells, while in contrast, REBOV infection resulted in a significant increase in ISG expression. Analysis of STAT1 and -2 phosphorylation following IFN treatment showed a significant reduction of STAT phosphorylation for MARV but not for ZEBOV and REBOV, indicating that different mechanisms might be involved in antagonizing IFN signaling pathways by the different filovirus species. Taken together, these studies showed a correlation between antagonism of type I IFN responses and filovirus virulence.

FIG. 1.

Global analysis of EBOV and MARV infection kinetics. Panel A, immunofluorescence analysis of Huh7 cells infected with ZEBOV, REBOV, or MARV at an MOI of 0.1 at 24 h and 48 h p.i. Panel B, two-dimensional agglomerative cluster matrix of genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression in a least one experiment. In the left panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, while black indicates no change in gene expression. In the right panel, genes whose regulation showed a P value of >0.01 are shown in gray.

FIG.2.

Control of host cell gene expression following EBOV and MARV infection. Panel A, Venn diagram showing the segregation of infection-regulated genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression at 24 h p.i. Hierarchical clustering matrix of the expression of genes in set I that were preferentially regulated by ZEBOV at 24 h, in set II that were commonly regulated by ZEBOV and MARV, in set III that were common to all viral infections, and in set IV that were restricted to REBOV. Panel B, Venn diagram showing the segregation of infection-regulated genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression at 48 h p.i. and the associated hierarchical clustering diagrams of genes in sets I to IV. Panel C, matrix showing the expression of selected inflammation- and apoptosis-related genes in ZEBOV-, REBOV-, and MARV-infected cells at 24 and 48 h p.i. In the left panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, while black indicates no change in gene expression. In the right panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel D, bar graphs comparing the expression of selected mRNAs as measured by quantitative real-time PCR and cDNA expression microarray analysis. The results are presented as the log ratio of mRNA abundance in infected relative to mock-infected cells.

FIG.2.

Control of host cell gene expression following EBOV and MARV infection. Panel A, Venn diagram showing the segregation of infection-regulated genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression at 24 h p.i. Hierarchical clustering matrix of the expression of genes in set I that were preferentially regulated by ZEBOV at 24 h, in set II that were commonly regulated by ZEBOV and MARV, in set III that were common to all viral infections, and in set IV that were restricted to REBOV. Panel B, Venn diagram showing the segregation of infection-regulated genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression at 48 h p.i. and the associated hierarchical clustering diagrams of genes in sets I to IV. Panel C, matrix showing the expression of selected inflammation- and apoptosis-related genes in ZEBOV-, REBOV-, and MARV-infected cells at 24 and 48 h p.i. In the left panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, while black indicates no change in gene expression. In the right panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel D, bar graphs comparing the expression of selected mRNAs as measured by quantitative real-time PCR and cDNA expression microarray analysis. The results are presented as the log ratio of mRNA abundance in infected relative to mock-infected cells.

FIG.2.

Control of host cell gene expression following EBOV and MARV infection. Panel A, Venn diagram showing the segregation of infection-regulated genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression at 24 h p.i. Hierarchical clustering matrix of the expression of genes in set I that were preferentially regulated by ZEBOV at 24 h, in set II that were commonly regulated by ZEBOV and MARV, in set III that were common to all viral infections, and in set IV that were restricted to REBOV. Panel B, Venn diagram showing the segregation of infection-regulated genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression at 48 h p.i. and the associated hierarchical clustering diagrams of genes in sets I to IV. Panel C, matrix showing the expression of selected inflammation- and apoptosis-related genes in ZEBOV-, REBOV-, and MARV-infected cells at 24 and 48 h p.i. In the left panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, while black indicates no change in gene expression. In the right panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel D, bar graphs comparing the expression of selected mRNAs as measured by quantitative real-time PCR and cDNA expression microarray analysis. The results are presented as the log ratio of mRNA abundance in infected relative to mock-infected cells.

FIG.2.

Control of host cell gene expression following EBOV and MARV infection. Panel A, Venn diagram showing the segregation of infection-regulated genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression at 24 h p.i. Hierarchical clustering matrix of the expression of genes in set I that were preferentially regulated by ZEBOV at 24 h, in set II that were commonly regulated by ZEBOV and MARV, in set III that were common to all viral infections, and in set IV that were restricted to REBOV. Panel B, Venn diagram showing the segregation of infection-regulated genes that showed a ≥2-fold (n = 4; P < 0.01) change in expression at 48 h p.i. and the associated hierarchical clustering diagrams of genes in sets I to IV. Panel C, matrix showing the expression of selected inflammation- and apoptosis-related genes in ZEBOV-, REBOV-, and MARV-infected cells at 24 and 48 h p.i. In the left panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, while black indicates no change in gene expression. In the right panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel D, bar graphs comparing the expression of selected mRNAs as measured by quantitative real-time PCR and cDNA expression microarray analysis. The results are presented as the log ratio of mRNA abundance in infected relative to mock-infected cells.

FIG.3.

Increased activation and expression of IFN-regulated genes by REBOV compared to ZEBOV and MARV. Panel A, scatter plots of the expression patterns of individual genes that were ≥2-fold (n = 4; P < 0.01) up-regulated by treatment with 100 IU/ml IFN-α-2b in mock-infected cells compared to their expression in ZEBOV-, REBOV-, and MARV-infected cells at 48 h. Panel B, hierarchical clustering matrix of the ISGs that were up-regulated at 24 h and 48 h p.i. with REBOV, ZEBOV, or MARV relative to mock-infected cells treated with IFN-α-2b. In the left panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, while black indicates no change in gene expression. In the right panel, genes whose regulation showed a P value of >0.01 are shown in gray.

FIG.3.

Increased activation and expression of IFN-regulated genes by REBOV compared to ZEBOV and MARV. Panel A, scatter plots of the expression patterns of individual genes that were ≥2-fold (n = 4; P < 0.01) up-regulated by treatment with 100 IU/ml IFN-α-2b in mock-infected cells compared to their expression in ZEBOV-, REBOV-, and MARV-infected cells at 48 h. Panel B, hierarchical clustering matrix of the ISGs that were up-regulated at 24 h and 48 h p.i. with REBOV, ZEBOV, or MARV relative to mock-infected cells treated with IFN-α-2b. In the left panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, while black indicates no change in gene expression. In the right panel, genes whose regulation showed a P value of >0.01 are shown in gray.

FIG.4.

Increased activation of type I IFN receptor responses by REBOV infection. Panel A, immunofluorescence analysis of Huh7 cells infected with ZEBOV, REBOV, or MARV at an MOI of 0.1 for 24 h and then treated with 100 IU/ml of IFN-α-2b for an additional 24 h. Panel B, quantification of infectious viral particles in culture supernatants of infected cells in the presence and absence of 100 IU/ml IFN-α-2b (for 24 h) expressed as the log₁₀ TCID₅₀/ml. Panel C, scatter plots comparing the expression of genes that were >1.5× (P < 0.01) induced in mock-infected cells treated with 100 IU/ml IFN-α-2b for 24 h and in MARV-, ZEBOV-, and REBOV-infected cells (24 h) treated IFN-α-2b for 24 h. Expression of MX1 RNA is indicated by arrows. Panel D, matrix of genes induced >1.5× (P < 0.01) in ZEBOV-, REBOV-, and MARV-infected cells treated with IFN. Panel E, hierarchical clustering matrix of selected genes that were preferentially induced in REBOV-infected cells following IFN-α-2b treatment. In the top panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, and black indicates no change in gene expression. In the bottom panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel F, Western blotting analysis showing expression of MX1 protein during ZEBOV, REBOV, and MARV infection in the absence and presence of IFN-α-2b.

FIG.4.

Increased activation of type I IFN receptor responses by REBOV infection. Panel A, immunofluorescence analysis of Huh7 cells infected with ZEBOV, REBOV, or MARV at an MOI of 0.1 for 24 h and then treated with 100 IU/ml of IFN-α-2b for an additional 24 h. Panel B, quantification of infectious viral particles in culture supernatants of infected cells in the presence and absence of 100 IU/ml IFN-α-2b (for 24 h) expressed as the log₁₀ TCID₅₀/ml. Panel C, scatter plots comparing the expression of genes that were >1.5× (P < 0.01) induced in mock-infected cells treated with 100 IU/ml IFN-α-2b for 24 h and in MARV-, ZEBOV-, and REBOV-infected cells (24 h) treated IFN-α-2b for 24 h. Expression of MX1 RNA is indicated by arrows. Panel D, matrix of genes induced >1.5× (P < 0.01) in ZEBOV-, REBOV-, and MARV-infected cells treated with IFN. Panel E, hierarchical clustering matrix of selected genes that were preferentially induced in REBOV-infected cells following IFN-α-2b treatment. In the top panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, and black indicates no change in gene expression. In the bottom panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel F, Western blotting analysis showing expression of MX1 protein during ZEBOV, REBOV, and MARV infection in the absence and presence of IFN-α-2b.

FIG.4.

Increased activation of type I IFN receptor responses by REBOV infection. Panel A, immunofluorescence analysis of Huh7 cells infected with ZEBOV, REBOV, or MARV at an MOI of 0.1 for 24 h and then treated with 100 IU/ml of IFN-α-2b for an additional 24 h. Panel B, quantification of infectious viral particles in culture supernatants of infected cells in the presence and absence of 100 IU/ml IFN-α-2b (for 24 h) expressed as the log₁₀ TCID₅₀/ml. Panel C, scatter plots comparing the expression of genes that were >1.5× (P < 0.01) induced in mock-infected cells treated with 100 IU/ml IFN-α-2b for 24 h and in MARV-, ZEBOV-, and REBOV-infected cells (24 h) treated IFN-α-2b for 24 h. Expression of MX1 RNA is indicated by arrows. Panel D, matrix of genes induced >1.5× (P < 0.01) in ZEBOV-, REBOV-, and MARV-infected cells treated with IFN. Panel E, hierarchical clustering matrix of selected genes that were preferentially induced in REBOV-infected cells following IFN-α-2b treatment. In the top panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, and black indicates no change in gene expression. In the bottom panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel F, Western blotting analysis showing expression of MX1 protein during ZEBOV, REBOV, and MARV infection in the absence and presence of IFN-α-2b.

FIG.4.

Increased activation of type I IFN receptor responses by REBOV infection. Panel A, immunofluorescence analysis of Huh7 cells infected with ZEBOV, REBOV, or MARV at an MOI of 0.1 for 24 h and then treated with 100 IU/ml of IFN-α-2b for an additional 24 h. Panel B, quantification of infectious viral particles in culture supernatants of infected cells in the presence and absence of 100 IU/ml IFN-α-2b (for 24 h) expressed as the log₁₀ TCID₅₀/ml. Panel C, scatter plots comparing the expression of genes that were >1.5× (P < 0.01) induced in mock-infected cells treated with 100 IU/ml IFN-α-2b for 24 h and in MARV-, ZEBOV-, and REBOV-infected cells (24 h) treated IFN-α-2b for 24 h. Expression of MX1 RNA is indicated by arrows. Panel D, matrix of genes induced >1.5× (P < 0.01) in ZEBOV-, REBOV-, and MARV-infected cells treated with IFN. Panel E, hierarchical clustering matrix of selected genes that were preferentially induced in REBOV-infected cells following IFN-α-2b treatment. In the top panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, and black indicates no change in gene expression. In the bottom panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel F, Western blotting analysis showing expression of MX1 protein during ZEBOV, REBOV, and MARV infection in the absence and presence of IFN-α-2b.

FIG.4.

Increased activation of type I IFN receptor responses by REBOV infection. Panel A, immunofluorescence analysis of Huh7 cells infected with ZEBOV, REBOV, or MARV at an MOI of 0.1 for 24 h and then treated with 100 IU/ml of IFN-α-2b for an additional 24 h. Panel B, quantification of infectious viral particles in culture supernatants of infected cells in the presence and absence of 100 IU/ml IFN-α-2b (for 24 h) expressed as the log₁₀ TCID₅₀/ml. Panel C, scatter plots comparing the expression of genes that were >1.5× (P < 0.01) induced in mock-infected cells treated with 100 IU/ml IFN-α-2b for 24 h and in MARV-, ZEBOV-, and REBOV-infected cells (24 h) treated IFN-α-2b for 24 h. Expression of MX1 RNA is indicated by arrows. Panel D, matrix of genes induced >1.5× (P < 0.01) in ZEBOV-, REBOV-, and MARV-infected cells treated with IFN. Panel E, hierarchical clustering matrix of selected genes that were preferentially induced in REBOV-infected cells following IFN-α-2b treatment. In the top panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, and black indicates no change in gene expression. In the bottom panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel F, Western blotting analysis showing expression of MX1 protein during ZEBOV, REBOV, and MARV infection in the absence and presence of IFN-α-2b.

FIG.4.

Increased activation of type I IFN receptor responses by REBOV infection. Panel A, immunofluorescence analysis of Huh7 cells infected with ZEBOV, REBOV, or MARV at an MOI of 0.1 for 24 h and then treated with 100 IU/ml of IFN-α-2b for an additional 24 h. Panel B, quantification of infectious viral particles in culture supernatants of infected cells in the presence and absence of 100 IU/ml IFN-α-2b (for 24 h) expressed as the log₁₀ TCID₅₀/ml. Panel C, scatter plots comparing the expression of genes that were >1.5× (P < 0.01) induced in mock-infected cells treated with 100 IU/ml IFN-α-2b for 24 h and in MARV-, ZEBOV-, and REBOV-infected cells (24 h) treated IFN-α-2b for 24 h. Expression of MX1 RNA is indicated by arrows. Panel D, matrix of genes induced >1.5× (P < 0.01) in ZEBOV-, REBOV-, and MARV-infected cells treated with IFN. Panel E, hierarchical clustering matrix of selected genes that were preferentially induced in REBOV-infected cells following IFN-α-2b treatment. In the top panel, genes shown in red were up-regulated and genes shown in green were down-regulated relative to uninfected Huh7 cells, and black indicates no change in gene expression. In the bottom panel, genes whose regulation showed a P value of >0.01 are shown in gray. Panel F, Western blotting analysis showing expression of MX1 protein during ZEBOV, REBOV, and MARV infection in the absence and presence of IFN-α-2b.

FIG.5.

Phosphorylation of STAT proteins in filovirus-infected cells following type I IFN treatment. Western blotting analysis was performed on equal-mass cell lysates obtained from mock-, SeV-, ZEBOV-, REBOV-, or MARV-infected cells at 24 h in the absence or presence of 100 IU/ml IFN-α-2b for 30 min, using antibodies specific for phosphorylated STAT1 or total STAT1 protein (A) or for phosphorylated STAT2 or total STAT2 protein (B).