Single-Particle Tracking and Positional Phenotyping Reveal Variant-Specific Early Checkpoints in SARS-CoV-2 Cell Entry.

Bulk assays of SARS-CoV-2 entry obscure individual virions and conflate binding with internalization. We developed a quantitative single-particle imaging assay that classifies fluorescent virus-like particles (VLPs) as surface, crossing, or internal on HEK293T-ACE2 cells, separating binding from internalization. Comparing G614 and Omicron BA.5 S protein variants, G614 showed higher binding and a larger internalized fraction at baseline. A trivalent anti-S protein aptamer reduced G614 binding and internalization but increased BA.5 internalization. These positional readouts expose variant-resolved early entry checkpoints and provide a simple platform to test how ligands shift binding and internalization.