The ongoing coronavirus disease 2019 (COVID-19) pandemic has significantly affected global healthcare systems and economies. Since the beginning of the pandemic, which was caused by the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), researchers have been continually investigating the replication mechanisms of this virus.
Study: NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids. Image Credit: marianstock / Shutterstock.com
SARS-CoV-2 infects humans through the interaction between its spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor of the host cell.
Previous studies have reported that SARS-CoV-2 primarily replicates in airway epithelial cells, which express high levels of the ACE2 receptor. As compared to the high level of ACE2 expression observed in the airways and lungs, the alveolar space exhibits a much lower level of ACE2 expression.
The primary function of the airway epithelium is to remove and neutralize harmful substances and pathogens present in the inhaled air. Club cells, for example, produce immunomodulatory club cell secretory proteins.
Comparatively, goblet cells secrete mucins, which form mucus on the internal surfaces of the respiratory tract that protect the underlying epithelium. Ciliated cells facilitate the movement of mucus through the respiratory tract.
Viral proteins have been detected in the airway epithelium and lung tissues of COVID-19 patients. Although ciliated cells appear to be natural targets of SARS-CoV-2, viral proteins have also been detected in basal and secretory cells in both in vivo and ex vivo infections.
Three-dimensional (3D) organoid models have been developed to mimic the complex cellularity of the human airway epithelium. These organoids consist of different types of cells grown in a 3D structure that mimics human organs.
Typically, these 3D organoid models are derived from pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or progenitor cells. In the context of SARS-CoV-2, iPSC- or ESC-derived human airway organoids (HAOs) are often used to study its replication pattern.
Since the original strain of SARS-CoV-2 was detected in 2019, it has undergone genomic mutations that have led to the emergence of several SARS-CoV-2 variants. These variants have been classified as variants of concern (VOCs) and variants of interest (VOIs) by the World Health Organization (WHO).
In a recent study published on the preprint server bioRxiv*, researchers developed a genetically modified airway organoid system, which overcomes challenges related to natural variations in ACE2 levels. This adult stem cell-derived undifferentiated HAO model offers a high infection rate without lengthy differentiation while also allowing researchers to analyze the effects of infection using genetically diverse SARS-CoV-2 variants.
In the current study, scientists applied a single-cell ribonucleic acid (RNA) sequencing method to decipher the cell-intrinsic response to SARS-CoV-2. To this end, they predicted that enhanced infection rates of SARS-CoV-2 variants could be observed in other ACE2-utilizing coronaviruses like hCOV-NL63 and SARS-CoV.
Transcriptional profiling of secretory goblet cells, club cells, and basal cells was also conducted. These cells exhibited common transcriptional changes, thus indicated by clustering following infection by SARS-CoV-2 variants.
The nuclear factor-κB inhibitor alpha (NFKBIA) gene was proposed to be effective in controlling SARS-CoV-2 infection by different variants. When the NFKBIA gene is highly induced, messenger ribonucleic acid (mRNA) levels are positively correlated with viral RNA levels.
A high level of the IκBα protein is expressed in cells infected with SARS-CoV-2. Despite upregulation of IκBα, the presence of NF-κB in the nucleus of infected cells, rather than healthy cells, indicates continual triggering of NF-κB signaling. This finding is consistent with a previous study that observed an incomplete feedback loop in NF-κB control during respiratory syncytial virus (RSV) infection,
This incomplete feedback loop in NF-κB could represent the ongoing race between SARS-CoV-2 and the host. Although a high rate of NFKBIA transcripts is present, continual upregulation of antiviral NF-κB signaling in the host due to the persistent presence of the virus affects the feedback loop. Additionally, continual synthesis of IκBα protein through constant degradation of the IκBα inhibitor affects viral replication.
The organoid model supports the phenomenon that overexpression of a mutant unphosphorylated IκBα protein is easily degraded. In this study, IκBα was reported to be a proviral factor that supports viral replication in the host.
The present study supports the previous observation that IκBα promotes viral infection by partially constraining the antiviral activities of NF-κB. In contrast to this observation, knockdown of p65 has demonstrated the positive effect of NF-κB signaling in SARS-CoV-2 infection.
The organoid model described in the current study could be a powerful tool for studying SARS-CoV-2 infection in primary airway cells. This model reduces donor-dependent variable infection rates and offers fully differentiated primary cell models. Importantly, it also helps understand the transcriptional reprogramming that occurs in progenitor cells of the airways in response to COVID-19 infection.
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.
- Simoneau, R. C., Chen, P., Xing, G. K., et al. (2022) NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids. bioRxiv. doi:10.1101/2022.08.02.502100