In a recent study published on the bioRxiv* preprint server, researchers assessed the impact of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry pathway on downstream cellular and viral processes.
Study: The entry pathway of SARS-CoV-2 affects a number of downstream viral and cellular processes. Image credit: PHOTOCREO Michal Bednarek/Shutterstock
Background
Despite variation in SARS-CoV-2 spike (S) proteins among variants, the entry step is critical in the SARS-CoV-2 life cycle. SARS-CoV-2 infection of host cells begins with cell attachment via C-type lectin receptors. The virus binds to cell membranes and the S protein directly binds to a primary receptor called angiotensin-converting enzyme 2 (ACE2).
Cleavage of the S protein exposes the fusion peptide, which facilitates host-virus membrane fusion, thereby creating a pore that allows virus release into host cells. This S cleavage occurs in the presence of human transmembrane protease serine 2 (TMPRSS2). TMPRSS2 expression levels therefore influence the mechanism used by the virus to enter ACE2-expressing cells.
About the research
In the current study, the researchers investigated the relationship of the entry pathway of SARS-CoV-2 to physiologically relevant downstream viral and host processes.
The team developed lentiviral transgenic delivery plasmids that encode mouse or human ACE2 proteins along with lentiviral transgenic delivery plasmids that encode either a catalytically inactive version (ΔHDS) or a functional TMPRSS2 protein. Plasmids were used to develop a panel of ectopically expressing cell lines, while Western blot analysis determined the expression of murine or human ACE2 in A549-A, A549-AT(ΔHDS), A549-AT, and A549-mAT cells.
Furthermore, the infection susceptibility of the engineered or parental cell lines was assessed using the virus progenitor (B.1) and SARS-CoV-2 B.1.617 and B.1.1.529 variants of concern (VOCs).
The sensitivity of the virus to inhibition of lung epithelial cell entry was assessed by performing authentic viral infections that were exposed to a TMPRSS2 inhibitor called camostat mesylate and a cathepsin B/L inhibitor called E-64d.
Additionally, the team assessed whether the incident plasma membrane surface entry could be artificially stimulated in TMPRSS2-deficient cells by performing infections under acidified conditions.
Results
The results of the study showed that the expression and auto-cleavage of TMPRSS2 resulted in two fragments in A549-T and A549-AT cells. The team observed accumulated full-length TMPRSS2 in A549-AT(ΔHDS) cells, which showed no serine protease activity and auto-cleavage. This confirmed the lack of endogenous expression of TMPRSS2 or ACE2 in the parental A549 cells.
Furthermore, B.1 replication was significantly increased in A549-AT cells with a modest boost in VOC. Enhancement in all three virus strains decreased to levels similar to or lower than those found in A549-A cells in the presence of inactive TMPRSS2. Notably, the accumulation of the B.1.1.529 nucleocapsid proteins was comparable in TMPRSS2 cells overexpressing either mouse or human ACE2.
However, accumulation was not similar for B.1 and B.1.617 variants, indicating a wide range of hosts that utilize mouse ACE2 for B.1.1.529. Furthermore, intracellular transcription and replication of B.1 and B.1.1.529 were remarkably increased in A549-AT cells than in A549-AT(ΔHDS) cells, which was not detected for the B.1.617 variant.
The team also noted that levels of virion secretion were significantly elevated in A549-AT cells for B.1, moderately elevated in A549-AT for B.1.617, and undetectable in supernatants from moderately elevated in A549-AT for B.1.617 and undetectable 210 in supernatants from A549-A and A549-AT for B.1.1.529. On the other hand, there was significant escape of B.1.1.529 in Vero E6 cells at lower levels than those of variants B.1 and B.1.617. This showed significant differences between lung epithelial cell tropism and TMPRSS2 dependence among different SARS-CoV-2 strains.
Furthermore, camostat mesylate modestly inhibited B.1 infection among A549-AT cells but not in A549-A cells. Of note, B.1.617 infection was not inhibited for either cell type. However, E-64d remarkably inhibited B.1.617 and B.1 infection of both cell types.
B.1 infection among A549-AT(ΔHDS) cells was further reduced under all conditions compared to A549-A cells, indicating that functionally inactive TMPRSS2 blocks ACE2 engagement through steric hindrance. This phenomenon was not detected for the B.1.617 variant, highlighting the potential variation in ACE2 engagement between strains.
Read coverage and average sequencing depth across viral genomes highlighted the variations in rates of early transcription and viral replication found between A549-AT and A549-A cells. This also showed highly conserved transcript profiles in the SARS-CoV-2 genomic profile, which confirmed the conservation of open reading frame (ORF) expression as well as genome replication kinetics between VOC and ancestral strains. Overall, the data show that expression of TMPRSS2 enhances the infection rate of SARS-CoV-2 strains B.1, B.1.617 and B.1.1.529.
Overall, the results of the study highlight the differences between SARS-CoV-2 B.1, B.1.617 and B.1.1.529 strains in terms of dependence on TMPRSS2 and their correlation with the downstream activation of immune responses after virus entry.
*Important message
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guiding clinical practice/health-related behavior or treated as established information.
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