In a recent study published on the preprint server bioRxiv*, researchers assessed the impact of the entry route of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on cellular and viral processes down
Study: Entry route of SARS-CoV-2 affects a number of downstream viral and cellular processes. Image credit: PHOTOCREO Michal Bednarek/Shutterstock
background
Despite the variation of SARS-CoV-2 spike (S) proteins between variants, the entry step is crucial 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 binds directly to the receptor primary called angiotensin converting enzyme 2 (ACE2).
Cleavage of the S protein exposes the fusion peptide, which facilitates fusion of host-viral membranes, thereby creating a pore that allows viral release into host cells. This S cleavage occurs in the presence of the human transmembrane serine 2 (TMPRSS2) protease. Therefore, TMPRSS2 expression levels influenced the mechanism used by the virus to enter ACE2-expressing cells.
About the study
In the present study, the researchers investigated the association of the SARS-CoV-2 entry route with physiologically relevant host and viral processes.
The team developed lentiviral transgene delivery plasmids encoding murine or human ACE2 proteins together with lentiviral transgene delivery plasmids encoding 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 murine or human ACE2 expression in A549-A, A549-AT (ΔHDS), A549 cells -AT and A549-mAT.
In addition, we assessed the susceptibility of engineered or parental cell lines to infection using the ancestral virus (B.1) and the SARS-CoV-2 variants of concern B.1.617 and B.1.1.529 (COV ).
The susceptibility of the virus to inhibition of entry into lung epithelial cells was assessed by performing authentic viral infections, which were exposed to a TMPRSS2 inhibitor called camostat mesylate and a cathepsin B/L inhibitor called E-64d .
Additionally, the team assessed whether the surface entry event at the plasma membrane could be artificially stimulated in TMPRSS2-deficient cells by performing infections under acidified conditions.
results
The results of the study showed that expression and self-cleavage of TMPRSS2 resulted in two fragments in A549-T and A549-AT cells. The team observed full-length TMPRSS2 accumulated in A549-AT(ΔHDS) cells that showed no serine protease activity or self-cleavage. This confirmed no endogenous expression of TMPRSS2 or ACE2 in parental A549 cells.
Furthermore, B.1 replication was significantly increased in A549-AT cells with a modest increase in VOCs. Enhancement of 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 B.1.1.529 nucleocapsid proteins was comparable to TMPRSS2 cells overexpressing murine or human ACE2.
However, accumulation was not similar for the B.1 and B.1.617 variants, indicating a wide range of hosts utilizing murine ACE2 for B.1.1.529. Furthermore, intracellular transcription and replication of B.1 and B.1.1.529 were markedly increased in A549-AT cells than in A549-AT cells (ΔHDS) which was not found by in variant B.1.617.
The team also noted that virion secretion rates were greatly increased in A549-AT cells for B.1, moderately increased in A549-AT for B.1,617, and undetectable in supernatants from a modest improvement in A549-AT for B.1.617 and undetectables. 210 in supernatants of A549-A and A549-AT for B.1.1.529. On the other hand, there was significant efflux of B.1.1.529 in Vero E6 cells at lower levels than that of the B.1 and B.1.617 variants. This showed significant differences between lung epithelial cell tropism and TMPRSS2 dependence between different SARS-CoV-2 strains.
Furthermore, camostat mesylate moderately inhibited B.1 infection among A549-AT cells, but not in A549-A cells. Notably, B.1.617 infection was not inhibited for either cell type. However, E-64d markedly inhibited B.1.617 and B.1 infection of both cell types.
B.1 infection among A549-AT (ΔHDS) cells was further decreased under all conditions compared to A549-A cells, indicating that functionally inactive TMPRSS2 blocked the commitment of ACE2 by steric hindrance. This phenomenon was not found for the B.1.617 variant, highlighting possible variations in ACE2 involvement between strains.
Average read coverage and sequencing depth across the viral genomes highlighted the variations in early transcription rates and viral replication found between A549-AT and A549-A cells. This also showed highly conserved transcription profiles across SARS-CoV-2 genomic profiling, which confirmed the conservation of open reading frame (ORF) expression as well as genome replication kinetics among VOC strains and ancestral Taken together, the data showed that TMPRSS2 expression enhanced the infection rates of SARS-CoV-2 strains B.1, B.1.617, and B.1.1.529.
Overall, the findings of the study highlighted the differences between SARS-CoV-2 strains B.1, B.1.617 and B.1.1.529 in terms of TMPRSS2 dependence and its correlation with activation downstream of immune responses following viral entry.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.