The COVID-19 pandemic is showing no signs of waning or extinction anytime soon. Now, a new study published on the preprint server bioRxiv* in August 2020 shows a new bioinformatics approach to studying the pathogenesis of this disease, by uncovering the differentially expressed genes (DEGs) and cell signaling pathways that cause the disease characteristics. This will help develop better therapies to counter the effects of these genes.
This transmission electron microscope image shows SARS-CoV-2—also known as 2019-nCoV, the virus that causes COVID-19—isolated from a patient in the U.S. Virus particles are shown emerging from the surface of cells cultured in the lab. The spikes on the outer edge of the virus particles give coronaviruses their name, crown-like. Image captured and colorized at NIAID's Rocky Mountain Laboratories (RML) in Hamilton, Montana. Credit: NIAID
The Viral Target and Disease Process
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus with a genome size that ranges between 26 and 32 kB. It has both structural proteins, including the spike glycoproteins, membrane proteins, envelope proteins and nucleocapsid, and non-structural proteins, including proteases. The infection of the host cell by this virus sets off an antiviral response. However, an unregulated immune response can contribute to the severe tissue damage seen in serious COVID-19 disease.
While a majority of COVID-19 patients are asymptomatic or show only mild symptoms, a significant minority develop severe lung inflammation, acute respiratory distress syndrome (ARDS), and some die. The disease involves not only ciliary dysfunction but also pro-inflammatory signaling pathways, leading to a macrophage activation syndrome, also called a cytokine storm. The pathogenesis of this severe deterioration in COVID-19 patients is unclear.
The virus primarily targets the human bronchial epithelium, which possesses the viral receptor, the angiotensin-converting enzyme (ACE) 2. In order to understand the pathogenesis of the disease, scientists have carried out microarray testing, but the small size of the sample limits these experiments.
The current study used a variety of technologies, including analysis of the GEO data, from the Gene Expression Omnibus to find the DEGs and the associated biological processes. The researchers worked on human bronchial organoids (hBOs).
Identification of DEGs
The mechanism of infection in the human bronchus was explored by comparing the transcriptional signature of these hBOs with uninfected controls. They found that the level of expression of 89 genes was different in these two types of cells. These DEGs comprised 59 up-regulated and 30 downregulated genes, in the hBOs, relative to the negative controls.
To understand the mechanism on SARS-CoV-2 infected human bronchus, the modular transcriptional signature of SARS-CoV-2 infected human bronchial organoids (hBOs) was compared to that of the uninfected controls. A total of 89 genes were identified to be differentially expressed in SARS-CoV-2 infected hBOs with the threshold of P<0.01. Among these DEGs, 59 were up-regulated and 30 down-regulated in SARS-CoV-2 infected hBOs compared with the negative controls.
Enrichment analysis of DEGs
The researchers then examined the DEGs using the KEGG pathway and the Gene Ontology (GO) categories for an enrichment analysis. KEGG is an integrated database resource which also has a computationally generated database in many categories such as metabolism, as well as other functions at the cellular and organism level. In their enrichment analysis, gene sets are interpreted by assigning genes to several predefined areas, based on their functional features.
The three top KEGG pathways led them to identify the top-ranking three components of the cells. These are the Cell fraction, Insoluble fraction, and Membrane fraction.
The Importance of Apoptosis
They then went on to identify the most important three biological processes, namely, Death, Cell Death, and Apoptosis. The researchers found that the death and apoptosis process was seen chiefly in the set of biological processes. The apoptosis pathway, with death signaling, is critical to clearing SARS-CoV-2 from hBOs. Genes such as XAF1, TNNF, and FAS are involved in T cell apoptosis in these patients.
Earlier research indicated that ciliary abnormalities in the bronchial epithelium are found in this disease. This, in turn, predisposes to secondary infections, apoptosis, and cell death. The infected airways show changes such as apoptosis and pericyte loss in the alveolar capillaries, as well as cell death caused by oxidative stress. As the disease progresses, the virus infects tracheal cells extensively, causing them to enter apoptosis and necrosis.
Enzyme Dysregulation in COVID-19
Thirdly, they picked out the top three molecular functions, which are the Enzyme Inhibitor activity, Peptidase Inhibitor activity, and Endopeptidase Inhibitor activity. The disease causes enzyme dysregulation in hBOs. Viral synthesis requires polyproteins to be translated from the viral RNA genome. Thus, the top three molecular processes in these infected cells are “Enzyme inhibitor activity”, “Peptidase inhibitor activity”, and “Endopeptidase inhibitor activity”.
The researchers comment, “These findings indicated that SARS-CoV-2 may inhibit the enzyme inhibitor activity in hBOs to create more polyproteins and viruses. Thus, hBOs may be a potential virus production base during COVID-19 diseases.”
Cytokine Binding and COVID-19 Severity
The researchers suggest that the main signaling pathways on KEGG analysis in SARS-CoV-2-infected hBOs are cytokine-cytokine receptor interaction, the P53 pathway, and apoptosis. Some of the cytokines that are secreted when the cell is exposed to an activator include IL-1, IL-6, and TNFα. These molecules interact with specific cell receptors on the cell surface to trigger a cell response. The SARS-CoV-2 also interacts with the surface receptor ACE2 via its spike protein, to achieve cell entry. This suggests that many cytokines bind to the surface of the infected cell and trigger inflammation simultaneously with the binding and entry of the virus. This could lead to the identification of these cytokines and suppression of such binding to reduce COVID-19 severity.
Central Role of the NF-κB pathway
The next step was to simulate a PPI network using a Cytoscape program. This protein-level PPI connects 84 nodes, with 30 interactions between the infected and uninfected hBOs. The functions of the most important gene modules of the infected vs uninfected hBOs were analyzed, and the top 10 biological pathways in terms of significance were identified. This showed the involvement of the NF-κB pathway in COVID-19.
This is a central pathway in inflammation since it is involved in the subsequent expression of pro-inflammatory genes, including cytokines, chemokines, and adhesion molecules. Its activation is known to underlie the induction of many genes in inflammatory conditions as well as regulating a multitude of signaling pathways, including STAT3, RGS12, and P53. The inhibition of this molecule could potentially reduce the severity of SARS-CoV-2 infection.
The study sums up: “NFKBIA, C3, and CCL20 may be key genes in SARS-CoV-2 infected hBOs. Our study suggested that “Cytokine-cytokine receptor interaction,” “P53 signaling pathway”, and “Apoptosis” were the main signaling pathways during SARS-CoV-2 infection. “ Appropriate intervention in these pathways could potentially turn around the course of the disease, these findings suggest.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.