Lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses

Abstract

The severity of respiratory viral infections is partially determined by the cellular response mounted by infected lung epithelial cells. Disease prevention and treatment is dependent on our understanding of the shared and unique responses elicited by diverse viruses, yet few studies compare host responses to viruses from different families while controlling other experimental parameters. Murine models are commonly used to study the pathogenesis of respiratory viral infections, and in vitro studies using murine cells provide mechanistic insight into the pathogenesis observed in vivo. We used microarray analysis to compare changes in gene expression of murine lung epithelial cells infected individually by three respiratory viruses causing mild (rhinovirus, RV1B), moderate (coronavirus, MHV-1), and severe (influenza A virus, PR8) disease in mice. RV1B infection caused numerous gene expression changes, but the differential effect peaked at 12 hours post-infection. PR8 altered an intermediate number of genes whose expression continued to change through 24 hours. MHV-1 had comparatively few effects on host gene expression. The viruses elicited highly overlapping responses in antiviral genes, though MHV-1 induced a lower type I interferon response than the other two viruses. Signature genes were identified for each virus and included host defense genes for PR8, tissue remodeling genes for RV1B, and transcription factors for MHV-1. Our comparative approach identified universal and specific transcriptional signatures of virus infection that can be used to distinguish shared and virus-specific mechanisms of pathogenesis in the respiratory tract.

Fig 1. Magnitude and timing of gene expression changes mediated by MHV, PR8, and RV.

The plots show the estimated log₂-fold change in expression relative to mock at 12 h vs. 24 h for each virus. Each point represents one gene; only those genes that differ significantly from mock (at either time point) are included. The solid black line is the best fit regression line with the gray shading showing the 95% confidence interval. The slope with confidence interval and R² are given in the inset legend. The dashed line illustrates the hypothesis that all changes in expression occur in the first 12 h (slope = 1); the dotted line shows the constant rate of change hypothesis (slope = 2). (A) MHV has small effects and most of the expression changes occur between 12 and 24 h (slope >> 2). (B) PR8 has larger effects than MHV and the changes approximate a constant rate of change across both 12 h intervals (slope ≈ 2). (C) RV also has large effects on gene expression and changes occur in the first 12 h with very little further change in the next 12 h (slope ≈ 1).

Fig 2. Numbers of genes with significantly altered expression upon viral infection.

Venn diagrams show the number of significantly (A) up- and (B) down-regulated genes compared to mock 24 h after infection. The proportion of genes that are uniquely significant for each virus is indicated in parentheses.

Fig 3. Patterns of gene expression changes mediated by viral infection.

(A) Genes differentially expressed in at least one viral infection at 24 h are plotted as log₂-fold change with each virus along a different axis. Signature genes, which have significantly larger effects in one virus compared to all other treatments, are colored: blue = PR8, red = RV, yellow = MHV. (B) The number of genes uniquely up- or down- regulated by each virus or pairs of viruses. The numbers along the center diagonal are the signature genes with boxes colored as in (A). The off-diagonal numbers are genes that have differential expression in two viruses compared to mock and the third virus, but are not significantly different from each other.

Fig 4. Differential expression of type I interferon-induced genes.

Genes with significantly up-regulated expression compared to mock at 24 h (see Fig 2) were used to query the Interferome v.2.01 database. The Venn diagram shows the number of shared and unique type I IFN-related genes that were up-regulated in each viral infection. The proportion of genes up-regulated in only one virus treatment are shown in parentheses. The genes represented in the Venn diagram were divided into functional groups and heat maps were generated using log₂-fold change values for each virus at 24 h compared to mock-inoculated controls. Heat maps of additional functional groups can be found in S3 Fig. Gene names are indicated to the right of each row and statistically significant values are outlined in black.

Fig 5. Differential expression of type I interferons and receptors.

The log₂-fold change compared to mock of up-regulated type I IFN cytokine and receptor genes. Gene names are shown to the right of each row and the color scale is the same as in Fig 4.