Rapid Discrimination of Single-Virus Entry Pathways via a Bioorthogonal Activatable Fluorescent Probe
Ai-Xin Ma1, Zhi-Gang Wang1, Yi-Fan Wang1
1State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Frontiers Science Centre For New Organic Matter, Research Centre for Analytical Sciences, College of Chemistry, School of Medicine, and Frontiers Science Center for Cell Responses, Nankai University, Tianjin, P.R. China.
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Viral transmembrane entry is a critical yet heterogeneous process that governs infectivity and pathogenesis. However, directly distinguishing virus attachment from internalization and resolving distinct entry mechanisms in real time at the single-virus level remains challenging. Here, we introduce a Bioorthogonal Click-Activatable Viral Labeling (CAVL) system for spatiotemporally resolved visualization of viral entry dynamics. This approach combines a membrane-anchored, tetrazine-quenched BODIPY probe (BDP-Tz-MAL) with viral particles dually labeled with trans-cyclooctene (TCO) on their surface and quantum dots (QDs) within their core. Virus-cell contact triggers a rapid inverse electron-demand Diels-Alder (iEDDA) reaction, generating a turn-on fluorescence signal exclusively during transmembrane entry. By integrating this activatable signal with a persistent internal QDs label, we achieve real-time discrimination of endocytic and non-endocytic entry pathways at single-particle resolution. The CAVL platform operates without genetic modification, applies to diverse enveloped and non-enveloped viruses, and provides a quantitative, background-suppressed strategy for elucidating viral entry mechanisms across broad biological contexts.
