Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis

Abstract

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

Fig.1. Host transcriptional responses induced by hMPV

(A) Kinetics of changes in genes expression. Total RNA extracted from uninfected and hMPV-infected A549 cells were hybridized to the HG-U133 plus 2.0 GeneChip Arrays, as described in Methods. The genes, filtered as significant in response to hMPV infection at p-value<=0.05 and with a fold change of two or above, compared to baseline (uninfected), are represented at various time post-infection. Fold change is calculated on average expression values for each time point compared to the baseline. (B) Clustering and heat map analysis of hMPV-regulated genes. The expression pattern of genes significantly altered in response to hMPV infection is represented as a hierarchical clustering, using UPGMA (Unweighted Pair-Group Method with Arithmetic mean) with Euclidean distance measure. The heat map is an intensity plot which represents the clusters within the dataset. The column dendrogram represent the cluster within the time post hMPV infection and row dendrogram represent the genes clusters with similar pattern within and across time points. The red color represents the up-regulation and green the down-regulation of gene expression over time. Treatment conditions are indicated at the bottom of the figure: C represents the uninfected cells; 6 h, 12 h, 24 h, 48 h and 72 h indicate the time points of hMPV infection.

Fig.1. Host transcriptional responses induced by hMPV

(A) Kinetics of changes in genes expression. Total RNA extracted from uninfected and hMPV-infected A549 cells were hybridized to the HG-U133 plus 2.0 GeneChip Arrays, as described in Methods. The genes, filtered as significant in response to hMPV infection at p-value<=0.05 and with a fold change of two or above, compared to baseline (uninfected), are represented at various time post-infection. Fold change is calculated on average expression values for each time point compared to the baseline. (B) Clustering and heat map analysis of hMPV-regulated genes. The expression pattern of genes significantly altered in response to hMPV infection is represented as a hierarchical clustering, using UPGMA (Unweighted Pair-Group Method with Arithmetic mean) with Euclidean distance measure. The heat map is an intensity plot which represents the clusters within the dataset. The column dendrogram represent the cluster within the time post hMPV infection and row dendrogram represent the genes clusters with similar pattern within and across time points. The red color represents the up-regulation and green the down-regulation of gene expression over time. Treatment conditions are indicated at the bottom of the figure: C represents the uninfected cells; 6 h, 12 h, 24 h, 48 h and 72 h indicate the time points of hMPV infection.

Fig.2. Gene ontology analysis of hMPV-induced transcriptional responses

The genes filtered as significant in response to hMPV infection at p-value<=0.05 and with a fold change of two or above, compared to baseline (uninfected), were analyzed using the gene ontology tool available in http://david.abcc.ncifcrf.gov and grouped using medium classification stringency. The representative groups with enrichment score of 1.5 or above and P value of 0.01 or less are presented in_(A) for the up-regulated genes and in (B) for the down-regulated genes.

Fig.2. Gene ontology analysis of hMPV-induced transcriptional responses

The genes filtered as significant in response to hMPV infection at p-value<=0.05 and with a fold change of two or above, compared to baseline (uninfected), were analyzed using the gene ontology tool available in http://david.abcc.ncifcrf.gov and grouped using medium classification stringency. The representative groups with enrichment score of 1.5 or above and P value of 0.01 or less are presented in_(A) for the up-regulated genes and in (B) for the down-regulated genes.

Fig.3. Changes in chemokine and cytokine expression after hMPV infection

A549 cells were infected with hMPV at MOI of 1 for various length of time. Total RNA was extracted from control and infected cells and subjected to a microarray analysis, as described in Methods. Induction of CXC chemokines (A), CC chemokines (B) and cytokines (C) genes is plotted as a function of time. Fold change represents the ratio of gene expression level of infected versus uninfected cells at the different time points.

Fig.4. Changes in the expression of interferons (IFN) and interferon stimulated genes (ISGs) after hMPV infection

A549 cells were infected with hMPV at MOI of 1 for various length of time. Total RNA was extracted from control and infected cells and subjected to a microarray analysis, as described in Methods. Induction of IFN-α (A), IFN-β and -λ (B), ISG (C) and TRIM (D) genes is plotted as a function of time. Fold change represents the ratio of gene expression level of infected versus uninfected cells at the different time points.

Fig.5. Induction of transcription factors after hMPV infection

A549 cells were infected with hMPV at MOI of 1 for various length of time. Total RNA was extracted from control and infected cells and subjected to a microarray analysis, as described in Methods. Induction of transcription factor genes is plotted as a function of time. Fold change represents the ratio of gene expression level of infected versus uninfected cells at the different time points.

Fig.6. Induction of TLRs, RNA helicases and MAPK signaling cascade molecules after hMPV infection

A549 cells were infected with hMPV at MOI of 1 for various length of time. Total RNA was extracted from control and infected cells and subjected to a microarray analysis, as described in Methods. Induction of TLR-related signaling molecules (A), RNA helicases (B), and signaling molecules related to the MAPK signaling cascade (C) is plotted as a function of time. Fold change represents the ratio of gene expression level of infected versus uninfected cells at the different time points. (D) Total cell lysates, prepared from A549 cells uninfected or infected with hMPV, MOI of 1, RSV for 6, 12 and 24 h were resolved on 10% SDS-PAGE and Western blot was performed using antibodies against RIG-I, MDA-5 and IKKε. Membranes were stripped and reprobed for β-actin as an internal control for protein integrity and loading.

Fig.7. Downregulation of antioxidant gene expression after hMPV infection

(A). Total RNA from control or infected A549 cells was subjected to a microarray analysis. The reduced induction of antioxidant genes is plotted as a function of time. Fold change represents the ratio of gene expression level of infected versus uninfected cells at the different time points. (B). A549 cells were infected with hMPV, MOI of 3, and harvested at 3, 6, 15 and 24 h post-infection to measure F2–isoprostanes. The figure is representative of two different experiments done in triplicate. (C). Total cell lysates, prepared from A549 cells uninfected or infected with hMPV, MOI of 3, for 15,24 and 48 h were resolved on 10% SDS-PAGE and Western blot was performed using antibodies against SOD 2, 3, catalase and GST. Membranes were stripped and reprobed for β-actin as an internal control for protein integrity and loading.