Transcriptional Profiling of SARS-CoV-2-Infected Calu-3 Cells Reveals Immune-Related Signaling Pathways

Abstract

The COVID-19 disease, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in late 2019 and rapidly spread worldwide, becoming a pandemic that infected millions of people and caused significant deaths. COVID-19 continues to be a major threat, and there is a need to deepen our understanding of the virus and its mechanisms of infection. To study the cellular responses to SARS-CoV-2 infection, we performed an RNA sequencing of infected vs. uninfected Calu-3 cells. Total RNA was extracted from infected (0.5 MOI) and control Calu-3 cells and converted to cDNA. Sequencing was performed, and the obtained reads were quality-analyzed and pre-processed. Differential expression was assessed with the EdgeR package, and functional enrichment was performed in EnrichR for Gene Ontology, KEGG pathways, and WikiPathways. A total of 1040 differentially expressed genes were found in infected vs. uninfected Calu-3 cells, of which 695 were up-regulated and 345 were down-regulated. Functional enrichment analyses revealed the predominant up-regulation of genes related to innate immune response, response to virus, inflammation, cell proliferation, and apoptosis. These transcriptional changes following SARS-CoV-2 infection may reflect a cellular response to the infection and help to elucidate COVID-19 pathogenesis, in addition to revealing potential biomarkers and drug targets.

Keywords: COVID-19; Calu-3 cells; RNA-seq; host-pathogen interaction; transcriptome.

Figure 1

Volcano plot showing differentially expressed genes (DEGs) in SARS-CoV-2-infected Calu-3 cells vs. control (uninfected) cells. The most expressive DEGs are identified. de: Differential expression. down: Down-regulated. no_sig: Non-significant. up: Up-regulated.

Figure 2

Heatmap of the top 20 DEGs in Calu-3 cells. Expression patterns of genes are compared between control (C1, C2, and C5) and infected (I1, I2, and I5) samples. For each gene, the relative values of gene expression are depicted in a blue-red scale, in which red tones are representative of higher expression, and blue tones, of lower expression. FPKM: Fragments per Kilobase Million.

Figure 3

Functional enrichment of up-regulated DEGs. Enriched terms for Gene Ontology’s (GO) biological process (A). Enriched terms for KEGG Pathways and WikiPathways (B).

Figure 4

Calu-3 cells 24 h after SARS-CoV-2 infection. The transcriptional features of the infected cells indicate a metabolic model with the activation of inflammatory and antiviral signaling, in addition to both apoptotic and cytoprotective/proliferative signaling. Up-regulated genes include a potential SARS-CoV-2 receptor (EPHA4), a chaperone (HSPA6), pro-inflammatory transcription factors (IRF3 and FOS), and inflammatory mediators (ACE, MMP17, IL6, SECTM1, and ISG15).