SARS-CoV-2 receptor ACE2 is co-expressed with genes related to transmembrane serine proteases, viral entry, immunity and cellular stress

Abstract

The COVID-19 pandemic resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in December 2019 in Wuhan in China has placed immense burden on national economies and global health. At present neither vaccination nor therapies are available. Here, we performed a meta-analysis of RNA-sequencing data from three studies employing human lung epithelial cells. Of these one focused on lung epithelial cells infected with SARS-CoV-2. We aimed at identifying genes co-expressed with angiotensin I converting enzyme 2 (ACE2) the human cell entry receptor of SARS-CoV-2, and unveiled several genes correlated or inversely correlated with high significance, among the most significant of these was the transmembrane serine protease 4 (TMPRSS4). Serine proteases are known to be involved in the infection process by priming the virus spike protein. Pathway analysis revealed virus infection amongst the most significantly correlated pathways. Gene Ontologies revealed regulation of viral life cycle, immune responses, pro-inflammatory responses- several interleukins such as IL6, IL1, IL20 and IL33, IFI16 regulating the interferon response to a virus, chemo-attraction of macrophages, and cellular stress resulting from activated Reactive Oxygen Species. We believe that this dataset will aid in a better understanding of the molecular mechanism(s) underlying COVID-19.

Figure 1

Lung epithelial cells (labeled “SARS004”) infected with SARS-CoV-2 cluster together with mock-infected lung epithelial cells but separated from all other lung epithelial cells.

Figure 2

Most significantly (Bonferoni-adjusted p < 1E−11) correlated (red to yellow in last column) or anti-correlated (green) genes with ACE2. The transmembrane serine protease 4 (TMPRSS4) is one of the most significantly correlated (r = 0.9142, p = 4.59E−20) with ACE2 suggesting a major role in priming the SARS-CoV2 spike protein. CXCL17 (r = 0.9273, p = 1.1E−21), ABCA12 (r = 0.9256, p = 1−92E−21) and ATP10B (r = 0.9193, p = 1.14E−20) had marginally higher correlation with ACE2 while another transmembrane protease TMPRS11E (r = 0.9121, p = 7.91E−20) had slightly lower correlation.

Figure 3

(a) Cluster analysis and gene expression heatmap of 72 most significantly (Bonferoni-adjusted p < 1E−11) correlated and anti.correlated genes with ACE2 shows close clustering of the serine protease TMPRSS4 with ACE2. (b) Heatmap of TMPRSS family members shows close clustering of TMPRSS11D/E and TMPRSS4 with ACE2 but also TMPRSS2 and TMPRSS13 have similar expression, particularly in SARS-CoV-2 infected samples. The red color bar indicates SARS-CoV-2, blue color bar control.

Figure 4

(a) The five most significantly overrepresented pathways correlated with ACE2 are Human papillomavirus infection, Bacterial invasion of epithelial cells, Endocytosis, Axon Guidance and Transcriptional mis-regulation in cancer. (b) The six most significantly overrepresented pathways anti-correlated with ACE2 are DNA replication, Metabolic pathways, Cell cycle, Fanconi anemia pathway, Mismatch repair and Homologous recombination. Many of these pathways are associated with DNA processing or repair. That these are down-regulated upon infection with SARS-CoV-2 is in line with reports about interferon and interferon stimulated genes (ISGs) inhibiting virus replication. This would be a defense against the attempts of the virus to recruit the host’s DNA repair and homologous recombination mechanisms as Gillespie et al. report.

Figure 5

GO analysis reflects virus entry and immune response involving ROS and inflammation. (a) Selected GOs (Biological processes) shows interaction of virus and host, cell-junction organization, endocytosis, reaction involving cytokine production. (b) Immunity-related GOs illustrate the immune response involving activation of myeloid cells and T-cells and interleukin-1 and other chemokine production. (c) GOs associated with ROS and inflammation demonstrate involvement of ROS and inflammation leading to apoptosis.

Figure 6

Protein interaction network of genes most significantly (Bonferoni-adjusted p < 1E−11) correlated and anti-correlated genes with ACE2 shows IFI16 (r = 0.8719), LIMA1 (r = 0.8955), CNNM3 (r = − 0.8732), HNF4A (r = − 0.8750), TRAF3IP2 (r = 0.8816), ASB2 (r = 0.8791) and FANCC (r = − 0.8682) as hub genes. Genes found as interactors in the BioGrid database are marked in red, the original geneset of ACE2-correlated genes is marked in green, hub genes and ACE2 have yellow shading.

Figure 7

Scheme of SARS-CoV-2 infection. The coronavirus SARS-CoV-2 docks at the receptor ACE2 on the membrane of the human epithelial cell. Transmembrane serine proteases TMPRSSx mediate SARS-CoV-2 cell entry via ACE2. TMPRSS2 was reported for this in the first SARS-CoV and by previous publication also for SARS-CoV-2 but we hypothesize that due to co-expression with ACE2, TMPRSS4 and other members of the TMPRSS family may well perform this task. We suggest that inhibitors of TMPRSS4 and other TMPRSS family members might have therapeutic potential. Upon entry into the cell, viral RNA is released, replicated and packaged again. Replication can be inhibited by interferon and interferon stimulated genes (ISG) what we also saw in negatively correlated replication pathways (e.g. DNA replication and homologous recombination). This indicates a healthy immune response and may be impaired in persons with a weak immune system due to age or disease. The packaged virus is released from the cell and can be attacked by macrophages chemo-attracted by CXCL17—or T-cells for which we found evidence for by GO analysis and by associated interleukins IL1 and IL7. It is tempting to speculate that the severity of the clinical manifestations such as the acute respiratory failure and also failure in other organs depends on the quality of the immune system decreasing with age or diseases such as diabetes. The involvement of ACE2 in the renin-angiotensin system as antagonist of ACE in regulating blood pressure via Angiotensin II (Ang-II), vasoconstriction, dilation and its protective role against lung injury are additional factors which correlate with age^,,.