A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells

Abstract

We developed a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy (Airyscan, STORM, and STED) and easily matches the spatial scale of single virus-cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in VeroE6 cells. Our results suggest that the variant of concern B.1.1.7, currently on the rise in several countries by a clear transmission advantage, in these cells outcompetes its ancestor B.1 in terms of a much faster kinetics of entry. Given the molecular scenario (entry by the late pathway and similar fraction of pre-cleaved S protein for B.1.1.7 and B.1), the faster entry of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike RBD with ACE2. Remarkably, we also observed directly the significant role of clathrin as mediator of late entry endocytosis, which had been previously suggested in analogy with other CoVs and from experiments on pseudotyped virus models. On overall, we believe that our fluroescence microscopy-based approach is valuable for future studies addressing of how SARS-CoV-2 and its variants interact with cells.

Digestive system infection by SARS‑CoV‑2: Entry mechanism, clinical symptoms and expression of major receptors (Review)

“…These mutations may change the structure of the protein, resulting in greater infectivity (23)(24)(25)(26). Storti et al (27) discovered that the B.1.1.7 strain is able to internalize faster and this accelerated internalization may be directly associated with the N501Y mutation of the S protein, which enhances the binding of RBd to AcE2. When the RBd gene is mutated, it may lead to an increase in the infection rate of SARS-coV-2 (28).…”

A Copper nanoparticles-based polymeric spray coating: Nanoshield against Sars-Cov-2

“…African green monkey kidney cells (Vero-E6) were grown as described previously. 12 A clinical isolate of SARS-CoV-2, kindly gifted from the San Raffaele Hospital (Milan, Italy) and propagated in Vero-E6 cells, was used for all experiments. All SARS-CoV-2 experiments were performed at a Biosafety Level 3 laboratory.…”

Virus morphology: Insights from super-resolution fluorescence microscopy

“…The assembly of viral virions occurs in the endoplasmic reticulum (ER) to Golgi intermediate compartment (ERGIC), followed by traffic to lysosomes and egress by Arl8b-dependent lysosomal exocytosis into the extracellular environment [ 49 ]. STORM imaging has been used to investigate the size of single virions during their entry and egress by focusing on the basal membrane plane of infected cells, and demonstrated the role of clathrin vesicles, rather than caveolin-1, as major carriers of the virus from the cell surface to the early endosome [ 50 ]. A combination of ExM and light sheet microscopy was used to investigate the expression kinetics and spatial arrangement of the four structural SARS-CoV-2 proteins, demonstrating an association of the N protein with ROs and allowing the visualization of virus-induced morphological changes of host cell structures [ 51 ].…”