Coxsackievirus B3 Infection of Human Neural Progenitor Cells Results in Distinct Expression Patterns of Innate Immune Genes

Abstract

Coxsackievirus B3 (CVB3), a member of Picornaviridae family, is an important human pathogen that causes a wide range of diseases, including myocarditis, pancreatitis, and meningitis. Although CVB3 has been well demonstrated to target murine neural progenitor cells (NPCs), gene expression profiles of CVB3-infected human NPCs (hNPCs) has not been fully explored. To characterize the molecular signatures and complexity of CVB3-mediated host cellular responses in hNPCs, we performed QuantSeq 3' mRNA sequencing. Increased expression levels of viral RNA sensors (RIG-I, MDA5) and interferon-stimulated genes, such as IFN-β, IP-10, ISG15, OAS1, OAS2, Mx2, were detected in response to CVB3 infection, while IFN-γ expression level was significantly downregulated in hNPCs. Consistent with the gene expression profile, CVB3 infection led to enhanced secretion of inflammatory cytokines and chemokines, such as interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). Furthermore, we show that type I interferon (IFN) treatment in hNPCs leads to significant attenuation of CVB3 RNA copy numbers, whereas, type II IFN (IFN-γ) treatment enhances CVB3 replication and upregulates suppressor of cytokine signaling 1/3 (SOCS) expression levels. Taken together, our results demonstrate the distinct molecular patterns of cellular responses to CVB3 infection in hNPCs and the pro-viral function of IFN-γ via the modulation of SOCS expression.

Keywords: Coxsackievirus B3 (CVB3); gene expression profiles; interferons; neuronal progenitor cells; suppressor of cytokine signaling.

Figure 1

Coxsackievirus B3 (CVB3) efficiently replicates in human neural progenitor cells (hNPCs) and causes minimal cytotoxicity. HeLa cells and hNPCs were infected with CVB3 at MOI of 1 or 5 for the indicated times. (A,C) RT-PCR was performed to measure CVB3 copy number and IFN-β mRNA levels. The expression of viral RNA copies was calculated in relation to the expression level of β-actin mRNA. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with mock-infected cells. (B,D) At the end of the incubation period, cell viability was determined using CellTiter Glo assays. Each value represents the mean ± SEM (n = 4).

Figure 2

QuantSeq analysis reveals differential expression patterns of host genes in CVB3-infected hNPCs. hNPCs were infected with CVB3 (MOI 5) and RNA was harvested at 4, 24, 48, and 72 h post infection (hpi). (A) Scatter plot shows the x-axis and y-axis indicating the expression levels of genes from mock-infected and virus-infected groups, respectively. Relatively highly expressed genes in the virus-infected groups (red) and mock-infected groups (green) are depicted. Black dots represent genes that were not classified as differentially expressed. The differentially expressed gene (DEG) count was identified by comparing mock- and virus-infected groups (fold change > 2) (B) Venn diagrams representing the overlap in DEG profiles of CVB3-infected cells. Numbers of upregulated genes (more than 100 folds) are indicated in red and downregulated genes are in blue. (C) Three-dimensional multi-dimensional scaling (3D-MDS) plot of QuantSeq datasets shows that 24, 48, and 72 hpi samples are clustered in locations different from the mock or 4 hpi samples.

Figure 3

Comprehensive analysis of gene expression profiles of CVB3-infected hNPCs reveals changes in innate immune response genes. (A) Gene ontology (B) Heatmap indicating the log₂ fold change values of differentially expressed genes (DEGs) involved in immune responses in response to CVB3 infection of hNPCs, as determined by QuantSeq analysis. Hierarchical clustering was performed to group genes based on similar expression profiles, as indicated by the dendrograms (left). Up- and downregulated DEGs are indicated in red and blue, respectively. Time points are indicated below the panels. (C) Interferon-stimulated genes–protein interaction networks of DEGs in 48 hpi samples were constructed using Cytoscape. Nodes represent proteins and edges represent interactions between two proteins. Fold changes in the gene expression of the virus-infected groups was overlaid onto the nodes, as indicated by the color key. (D) GG analysis of 48 hpi sample resulted in a list of DEGs involved in RIG-I like receptor signaling pathways. Red, green, and white colors indicate significantly increased, significantly decreased, and unchanged gene expression in mock vs. CVB3-infected cells. DEGs were labelled on the map of RIG-I like receptor signaling pathways obtained from KEGG database with official permission and guidance [26,27,28].

Figure 4

Interferon signaling and inflammatory responses are induced in CVB3-infected hNPCs. (A) Quantitative RT-PCR analysis was performed to measure RIG-I, MDA5, IFN-β, IP-10, ISG-15, OAS1, OAS2, and Mx2 transcript levels following CVB3 infection (MOI 5) in hNPCs. * p < 0.05; ** p < 0.01; *** p < 0.001, compared with mock-infected cells. (B) The concentration of secreted inflammatory cytokines/chemokines (IL-6, IL-8, MCP-1) in the cultured media was determined by ELISA. * p < 0.05; ** p < 0.01; *** p < 0.001 versus mock-infected control cells (C) The protein levels of RIG-I, MDA5, MAVS, and β-actin were measured by western blotting.

Figure 5

Type II interferon (IFN) signaling results in enhanced viral replication via upregulation of suppressor of cytokine signaling (SOCS)1 and SOCS3. (A) Recombinant human IFN-α (10 ng/mL), IFN-β (10 ng/mL), IFN-γ (10 ng/mL), IFN-λ1 (100 ng/mL), and IFN-λ2 (100 ng/mL) were added to hNPCs. The next day, cells were infected with CVB3 (MOI 5) and viral RNA expression was measured by RT-PCR. Data are shown as means ± SEM of three independent experiments. (B–D) hNPCs were treated with various doses of IFN-γ overnight, followed by CVB3 infection (MOI 5). (B) CVB3 copy number was determined (C) The protein levels of IFN-γ downstream signaling pathways, such as pSTAT1/STAT1, pSTAT3/STAT3, and interferon regulatory factor 1 (IRF1) were measured by western blotting. (D) RT-PCR was performed to measure relative SOCS1 and SOCS3 mRNA levels. * p < 0.05; ** p < 0.01; *** p < 0.001 versus mock-infected control cells. (E,F) HeLa cells were transfected with empty vector (EV), SOCS1-MYC tag, or SOCS3-MYC tag encoding plasmids for 24 h. The next day, cells were infected with CVB3 (MOI 1) for 8 hpi. Transfection efficiency was confirmed by measuring each gene expression level. Changes in the transcriptional expression of CVB3 vRNA were measured using RT-PCR. Transcript expression levels were calculated in relation to the expression level of β-actin and expressed as a fold-change in comparison to the expression level in EV-transfected control cells. * p < 0.05 versus mock-infected EV-transfected cells. (G,H) HeLa cells were transfected with EV, MYC-SOCS1, or MYC-SOCS3 encoding plasmids for 24 h and infected with CVB3 for 8 h. Cells were immunostained with anti-Coxsackievirus B3 antibody and anti-MYC antibody to detect SOCS proteins. CVB3 is indicated in red and cell nuclei are stained blue. The images are representative of three independent experiments. Scale bar represents 20 μm. CVB3-infected cells were counted and presented as % in the graph.