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Cytokine systems approach demonstrates differences in innate and pro-inflammatory host responses between genetically distinct MERS-CoV isolates.

Selinger C, Tisoncik-Go J, Menachery VD, Agnihothram S, Law GL, Chang J, Kelly SM, Sova P, Baric RS, Katze MG - BMC Genomics (2014)

Bottom Line: Using topological techniques, including persistence homology and filtered clustering, we performed a comparative transcriptional analysis of human Calu-3 cell host responses to the different MERS-CoV strains, with MERS-CoV Eng 1 inducing early kinetic changes, between 3 and 12 hours post infection, compared to MERS-CoV SA 1.Through our genomics-based approach, we found topological differences in the kinetics and magnitude of the host response to MERS-CoV SA 1 and MERS-CoV Eng 1, with differential expression of innate immune and pro-inflammatory responsive genes as a result of IFN, TNF and IL-1α signaling.Predicted activation for STAT3 mediating gene expression relevant for epithelial cell-to-cell adherens and junction signaling in MERS-CoV Eng 1 infection suggest that these transcriptional differences may be the result of amino acid differences in viral proteins known to modulate innate immunity during MERS-CoV infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington, USA. csel@uw.edu.

ABSTRACT

Background: The recent emergence of a novel coronavirus in the Middle East (designated MERS-CoV) is a reminder of the zoonotic and pathogenic potential of emerging coronaviruses in humans. Clinical features of Middle East respiratory syndrome (MERS) include atypical pneumonia and progressive respiratory failure that is highly reminiscent of severe acute respiratory syndrome (SARS) caused by SARS-CoV. The host response is a key component of highly pathogenic respiratory virus infection. Here, we computationally analyzed gene expression changes in a human airway epithelial cell line infected with two genetically distinct MERS-CoV strains obtained from human patients, MERS-CoV SA 1 and MERS-CoV Eng 1.

Results: Using topological techniques, including persistence homology and filtered clustering, we performed a comparative transcriptional analysis of human Calu-3 cell host responses to the different MERS-CoV strains, with MERS-CoV Eng 1 inducing early kinetic changes, between 3 and 12 hours post infection, compared to MERS-CoV SA 1. Robust transcriptional changes distinguished the two MERS-CoV strains predominantly at the late time points. Combining statistical analysis of infection and cytokine-stimulated Calu-3 transcriptomics, we identified differential innate responses, including up-regulation of extracellular remodeling genes following MERS-CoV Eng 1 infection and differential pro-inflammatory responses.

Conclusions: Through our genomics-based approach, we found topological differences in the kinetics and magnitude of the host response to MERS-CoV SA 1 and MERS-CoV Eng 1, with differential expression of innate immune and pro-inflammatory responsive genes as a result of IFN, TNF and IL-1α signaling. Predicted activation for STAT3 mediating gene expression relevant for epithelial cell-to-cell adherens and junction signaling in MERS-CoV Eng 1 infection suggest that these transcriptional differences may be the result of amino acid differences in viral proteins known to modulate innate immunity during MERS-CoV infection.

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Differentially expressed genes following MERS-CoV SA 1 and MERS-CoV Eng 1 infections. A. Statistically significant DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 were identified at 3, 7, 12, 18 and 24 hpi (absolute log2 FC > 1, FDR-corrected p-value < 0.05). The bargraph shows the number of up-regulated and down-regulated DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 at each time point. B. Heatmap of differentially expressed genes following MERS-CoV SA 1 and MERS-CoV Eng 1 infection shows more than four hundred genes uniquely expressed in MERS-CoV Eng 1 infected cells at early time points (between 3 and 12 hpi), with differential expression criteria of absolute log2 FC > 1 against time- and dataset-matched mocks, FDR-corrected p-value < 0.05).
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Fig3: Differentially expressed genes following MERS-CoV SA 1 and MERS-CoV Eng 1 infections. A. Statistically significant DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 were identified at 3, 7, 12, 18 and 24 hpi (absolute log2 FC > 1, FDR-corrected p-value < 0.05). The bargraph shows the number of up-regulated and down-regulated DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 at each time point. B. Heatmap of differentially expressed genes following MERS-CoV SA 1 and MERS-CoV Eng 1 infection shows more than four hundred genes uniquely expressed in MERS-CoV Eng 1 infected cells at early time points (between 3 and 12 hpi), with differential expression criteria of absolute log2 FC > 1 against time- and dataset-matched mocks, FDR-corrected p-value < 0.05).

Mentions: The greatest number of gene expression differences between MERS-CoV SA 1 and MERS-CoV Eng 1 were induced at the late time points, with 2160 genes and 2611 genes differentially expressed (DE) at 18 and 24 hpi, respectively (Figure 3A). There was a total of 4861 DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 in at least one time point (herein known as contrasting genes). In comparison to MERS-CoV SA 1, there was an earlier host response to MERS-CoV Eng 1 at 7 and 12 hpi. The accelerated Calu-3 response to MERS-CoV Eng 1 may be the result of the difference in kinetics of viral gRNA replication, with MERS-CoV Eng 1 more efficiently replicating at 3 and 7 hpi compared to MERS-CoV SA 1 (Figure 1A). Alternatively, the delay in the host response to MERS-CoV SA 1 may be due to the virus more efficiently evading innate immune responses that leads to enhanced viral replication compared to MERS-CoV Eng 1 (Additional file 2: Table S4). Between the two viruses there are amino acid differences in ORF4a and PLpro, viral proteins known to modulate the innate immune response during MERS infection [5–7]. We further examined host gene expression at the early time-points (3, 7 and 12 hpi) and found significant enrichment of genes associated with the STAT3 pathway. STAT3 pathway genes CDC25A, MYC, SOCS3 and SOCS4 were more strongly induced by MERS-CoV Eng 1 compared to MERS-CoV SA 1, particularly at 7 hpi (Figure 3B). Increased SOCS gene expression and decreased expression of PIM1 gene in response to MERS-CoV Eng1 indicated decreased STAT3 activity and possibly differential induction of apoptosis-related pathways. In a direct virus comparison, differential expression of pro-apoptotic BID, BAX, and BIM genes was observed at the early time-points and by 24 hpi there was extensive cytopathic effects caused by both viral infections that was likely the result of caspase-dependent apoptosis, as previously shown for MERS infection [20]. Of the 4861 contrasting genes, 2653 genes were also DE against time- and data set-matched mocks. Hierarchical clustering of the 4861 DE genes resulted in distinct gene clusters, with striking expression pattern contrasts between MERS-CoV SA 1 and MERS-CoV Eng 1 at 18 and 24 hpi. As shown in Table 2, functional analysis of the five most prominent clusters with contrasting gene expression revealed enrichment of integrin linked kinase (ILK) signaling and epithelial adherens junction signaling pathways, glutathione metabolism, and interferon and pro-inflammatory signaling pathways. Genes related to glutathione metabolism included GSTM1 and GSTM3, which were strongly downregulated in response to both viruses. ISGs, IFIT1 and IFIT3, were highly induced in response to both MERS-CoV Eng 1 and MERS-CoV SA 1, with pronounced early up-regulation specifically in response to MERS-CoV Eng 1. Genes associated with ILK signaling and epithelial adherens junction signaling pathways were strongly downregulated in response to MERS-CoV SA 1, whereas MERS-CoV Eng 1 predominantly up-regulated expression of these genes at the late time points (Additional file 3: Figure S1). Within the highly enriched pathways, cellular genes including PVRL1, RHOF and CREBBP with highest expression contrasts between the two infections were chosen for confirmation by qRT-PCR (Additional file 4: Figure S2). Pro-inflammatory CSF3 gene was found more highly induced by MERS-CoV SA 1 (log ratios in MERS-CoV SA1 and MERS-CoV Eng1 respectively: 2.2 and 0.5), whereas CCL5 gene was more highly induced by MERS-CoV Eng 1 (log ratios in MERS-CoV SA1 and MERS-CoV Eng1 respectively: 0.71 and 2.2). Examination of the aforementioned contrasting genes showed that only a small number of those were already differentially expressed at the early time-points. For example, expression of RHOF gene in response to MERS-CoV Eng 1 was increased 4-fold relative to mock at 12 hpi, whereas MERS-CoV SA 1 did not induce RHOF gene expression at this time-point. We therefore focused on differences in the host response mainly at the later time-points that had the highest number of contrasting genes.Figure 3


Cytokine systems approach demonstrates differences in innate and pro-inflammatory host responses between genetically distinct MERS-CoV isolates.

Selinger C, Tisoncik-Go J, Menachery VD, Agnihothram S, Law GL, Chang J, Kelly SM, Sova P, Baric RS, Katze MG - BMC Genomics (2014)

Differentially expressed genes following MERS-CoV SA 1 and MERS-CoV Eng 1 infections. A. Statistically significant DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 were identified at 3, 7, 12, 18 and 24 hpi (absolute log2 FC > 1, FDR-corrected p-value < 0.05). The bargraph shows the number of up-regulated and down-regulated DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 at each time point. B. Heatmap of differentially expressed genes following MERS-CoV SA 1 and MERS-CoV Eng 1 infection shows more than four hundred genes uniquely expressed in MERS-CoV Eng 1 infected cells at early time points (between 3 and 12 hpi), with differential expression criteria of absolute log2 FC > 1 against time- and dataset-matched mocks, FDR-corrected p-value < 0.05).
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Related In: Results  -  Collection

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Fig3: Differentially expressed genes following MERS-CoV SA 1 and MERS-CoV Eng 1 infections. A. Statistically significant DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 were identified at 3, 7, 12, 18 and 24 hpi (absolute log2 FC > 1, FDR-corrected p-value < 0.05). The bargraph shows the number of up-regulated and down-regulated DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 at each time point. B. Heatmap of differentially expressed genes following MERS-CoV SA 1 and MERS-CoV Eng 1 infection shows more than four hundred genes uniquely expressed in MERS-CoV Eng 1 infected cells at early time points (between 3 and 12 hpi), with differential expression criteria of absolute log2 FC > 1 against time- and dataset-matched mocks, FDR-corrected p-value < 0.05).
Mentions: The greatest number of gene expression differences between MERS-CoV SA 1 and MERS-CoV Eng 1 were induced at the late time points, with 2160 genes and 2611 genes differentially expressed (DE) at 18 and 24 hpi, respectively (Figure 3A). There was a total of 4861 DE genes between MERS-CoV SA 1 and MERS-CoV Eng 1 in at least one time point (herein known as contrasting genes). In comparison to MERS-CoV SA 1, there was an earlier host response to MERS-CoV Eng 1 at 7 and 12 hpi. The accelerated Calu-3 response to MERS-CoV Eng 1 may be the result of the difference in kinetics of viral gRNA replication, with MERS-CoV Eng 1 more efficiently replicating at 3 and 7 hpi compared to MERS-CoV SA 1 (Figure 1A). Alternatively, the delay in the host response to MERS-CoV SA 1 may be due to the virus more efficiently evading innate immune responses that leads to enhanced viral replication compared to MERS-CoV Eng 1 (Additional file 2: Table S4). Between the two viruses there are amino acid differences in ORF4a and PLpro, viral proteins known to modulate the innate immune response during MERS infection [5–7]. We further examined host gene expression at the early time-points (3, 7 and 12 hpi) and found significant enrichment of genes associated with the STAT3 pathway. STAT3 pathway genes CDC25A, MYC, SOCS3 and SOCS4 were more strongly induced by MERS-CoV Eng 1 compared to MERS-CoV SA 1, particularly at 7 hpi (Figure 3B). Increased SOCS gene expression and decreased expression of PIM1 gene in response to MERS-CoV Eng1 indicated decreased STAT3 activity and possibly differential induction of apoptosis-related pathways. In a direct virus comparison, differential expression of pro-apoptotic BID, BAX, and BIM genes was observed at the early time-points and by 24 hpi there was extensive cytopathic effects caused by both viral infections that was likely the result of caspase-dependent apoptosis, as previously shown for MERS infection [20]. Of the 4861 contrasting genes, 2653 genes were also DE against time- and data set-matched mocks. Hierarchical clustering of the 4861 DE genes resulted in distinct gene clusters, with striking expression pattern contrasts between MERS-CoV SA 1 and MERS-CoV Eng 1 at 18 and 24 hpi. As shown in Table 2, functional analysis of the five most prominent clusters with contrasting gene expression revealed enrichment of integrin linked kinase (ILK) signaling and epithelial adherens junction signaling pathways, glutathione metabolism, and interferon and pro-inflammatory signaling pathways. Genes related to glutathione metabolism included GSTM1 and GSTM3, which were strongly downregulated in response to both viruses. ISGs, IFIT1 and IFIT3, were highly induced in response to both MERS-CoV Eng 1 and MERS-CoV SA 1, with pronounced early up-regulation specifically in response to MERS-CoV Eng 1. Genes associated with ILK signaling and epithelial adherens junction signaling pathways were strongly downregulated in response to MERS-CoV SA 1, whereas MERS-CoV Eng 1 predominantly up-regulated expression of these genes at the late time points (Additional file 3: Figure S1). Within the highly enriched pathways, cellular genes including PVRL1, RHOF and CREBBP with highest expression contrasts between the two infections were chosen for confirmation by qRT-PCR (Additional file 4: Figure S2). Pro-inflammatory CSF3 gene was found more highly induced by MERS-CoV SA 1 (log ratios in MERS-CoV SA1 and MERS-CoV Eng1 respectively: 2.2 and 0.5), whereas CCL5 gene was more highly induced by MERS-CoV Eng 1 (log ratios in MERS-CoV SA1 and MERS-CoV Eng1 respectively: 0.71 and 2.2). Examination of the aforementioned contrasting genes showed that only a small number of those were already differentially expressed at the early time-points. For example, expression of RHOF gene in response to MERS-CoV Eng 1 was increased 4-fold relative to mock at 12 hpi, whereas MERS-CoV SA 1 did not induce RHOF gene expression at this time-point. We therefore focused on differences in the host response mainly at the later time-points that had the highest number of contrasting genes.Figure 3

Bottom Line: Using topological techniques, including persistence homology and filtered clustering, we performed a comparative transcriptional analysis of human Calu-3 cell host responses to the different MERS-CoV strains, with MERS-CoV Eng 1 inducing early kinetic changes, between 3 and 12 hours post infection, compared to MERS-CoV SA 1.Through our genomics-based approach, we found topological differences in the kinetics and magnitude of the host response to MERS-CoV SA 1 and MERS-CoV Eng 1, with differential expression of innate immune and pro-inflammatory responsive genes as a result of IFN, TNF and IL-1α signaling.Predicted activation for STAT3 mediating gene expression relevant for epithelial cell-to-cell adherens and junction signaling in MERS-CoV Eng 1 infection suggest that these transcriptional differences may be the result of amino acid differences in viral proteins known to modulate innate immunity during MERS-CoV infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington, USA. csel@uw.edu.

ABSTRACT

Background: The recent emergence of a novel coronavirus in the Middle East (designated MERS-CoV) is a reminder of the zoonotic and pathogenic potential of emerging coronaviruses in humans. Clinical features of Middle East respiratory syndrome (MERS) include atypical pneumonia and progressive respiratory failure that is highly reminiscent of severe acute respiratory syndrome (SARS) caused by SARS-CoV. The host response is a key component of highly pathogenic respiratory virus infection. Here, we computationally analyzed gene expression changes in a human airway epithelial cell line infected with two genetically distinct MERS-CoV strains obtained from human patients, MERS-CoV SA 1 and MERS-CoV Eng 1.

Results: Using topological techniques, including persistence homology and filtered clustering, we performed a comparative transcriptional analysis of human Calu-3 cell host responses to the different MERS-CoV strains, with MERS-CoV Eng 1 inducing early kinetic changes, between 3 and 12 hours post infection, compared to MERS-CoV SA 1. Robust transcriptional changes distinguished the two MERS-CoV strains predominantly at the late time points. Combining statistical analysis of infection and cytokine-stimulated Calu-3 transcriptomics, we identified differential innate responses, including up-regulation of extracellular remodeling genes following MERS-CoV Eng 1 infection and differential pro-inflammatory responses.

Conclusions: Through our genomics-based approach, we found topological differences in the kinetics and magnitude of the host response to MERS-CoV SA 1 and MERS-CoV Eng 1, with differential expression of innate immune and pro-inflammatory responsive genes as a result of IFN, TNF and IL-1α signaling. Predicted activation for STAT3 mediating gene expression relevant for epithelial cell-to-cell adherens and junction signaling in MERS-CoV Eng 1 infection suggest that these transcriptional differences may be the result of amino acid differences in viral proteins known to modulate innate immunity during MERS-CoV infection.

Show MeSH
Related in: MedlinePlus