Understanding Host Receptor Roles in Enveloped Viral Entry by Learning From SARS-CoV-2 and ACE2 65 views

Enveloped viruses exploit existing biochemical pathways for their own benefit to establish infection. Yet despite consisting of a diverse group of pathogens, they demonstrate remarkably similar strategies related to the entry process of infection. This process usually involves binding to receptors expressed on the surface of the plasma membrane of host cells and undergoing a series of sequential biochemical steps, which ultimately result in viral-host membrane fusion. The entry process for SARS-CoV-2 is mediated by the spike glycoprotein and the canonical entry point involves binding to the cell-surface receptor, ACE2. Subsequent activation of the spike glycoprotein via a suitable protease triggers viral-host membrane fusion. We have provided evidence demonstrating that SARS-CoV-2 adopts an opportunistic model of infection, wherein entry can proceed through different biochemical routes and different proteases provide sufficient activity to trigger membrane fusion. In addition, we have also shown that SARS-CoV-2 fusion can proceed even in the absence of ACE2 using synthetic membranes, as long as both i) appropriate attachment factors and ii) suitable protease are present. Spike engagement with soluble receptor removes at least one of two kinetic barriers, but failed to recapitulate faster kinetics observed in reconstituted biological membranes, hinting that there remained an unidentified factor influencing the entry process for SARS-CoV-2. Here, we attempt to identify this contribution from the plasma membrane by probing how stoichiometric arrangement of the ACE2 receptor affects binding and subsequent membrane fusion for SARS-CoV-2. To accomplish this, we utilized single molecule photobleaching measurements in plasma membrane vesicles (PMVs) from a mutant cell line expressing ACE2 tagged with GFP and derived a correlative model to determine copy numbers of ACE2. We combine this with single virus fusion observations of SARS-CoV-2 and leverage a new configuration of a powerful microfluidic-based assay. Advancing the understanding of SARS-CoV-2 entry, we improve our capability to probe the viral entry process and interrogate a pathogen that has adapted an infection model which is primed for evolutionary plasticity. Taken together, these contributions have expanded upon recent methods to systematically probe the entry requirements for enveloped viruses where there is a suspicion of impactful interplay between host receptor stoichiometry and viral glycoprotein engagement.

PHD (Doctor of Philosophy)

SARS-CoV-2; viral entry; ACE2; viral fusion; enveloped viruses; infectious disease; spike protein

English

2026-05-01