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While actin is a central regulator of viral entry, direct observations of actin-aided trafficking have been restricted to membrane protrusions on glass surfaces given the limitations of conventional methods. Here, high-speed 3D Tracking and Imaging microscopy (3D-TrIm) is integrated with highly photostable StayGold-labeled SARS-CoV-2 virus-like particles to capture long-term, high-resolution single-virus trajectories in live cells. This approach revealed distinct regimes of viral dynamics, including extracellular diffusion, protrusion-based surfing, and an unreported linear trafficking mode along the plasma membrane that precedes viral internalization. This work demonstrates that this membrane trafficking is actin-driven and positively correlated with ACE2 expression. These findings reveal new actin exploitation by viruses and demonstrate the utility of 3D-TrIm for dissecting dynamic virus\u2013cell interactions at high spatiotemporal resolution.<\/jats:p>","DOI":"10.64898\/2026.04.03.716319","type":"posted-content","created":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T03:30:17Z","timestamp":1775532617000},"source":"Crossref","is-referenced-by-count":1,"title":["High-speed 3D single-virus tracking reveals actin-aided viral trafficking of SARS-CoV-2 on the plasma membrane"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1758-6215","authenticated-orcid":false,"given":"Yuxin","family":"Lin","sequence":"first","affiliation":[{"name":"Department of Chemistry, Duke University; Durham, North Carolina 27708, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-6410-2014","authenticated-orcid":false,"given":"Xinrui","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Duke University; 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