The Delta variant SARS-CoV-2 spike protein uniquely promotes aggregation of pseudotyped viral particles

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.2, exhibit faster initial infection with a higher viral load than prior variants, and pseudotyped particles bearing the SARS-CoV-2 Delta variant spike protein induce a faster initial infection rate of target cells compared to those bearing other SARS-CoV-2 variant spikes. Here, we show that pseudotyped particles bearing the Delta variant spike form unique aggregates, as evidenced by negative stain and cryogenic electron microscopy (EM), flow cytometry, and nanoparticle tracking analysis. Viral particles pseudotyped with other SARS-CoV-2 spike variants do not show aggregation by any of these criteria. The contribution to infection kinetics of the Delta spikes unique property to aggregate is discussed with respect to recent evidence for collective infection by other viruses. Irrespective of this intriguing possibility, spike-dependent aggregation is a new functional parameter of spike-expressing viral particles to evaluate in future spike protein variants.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.