Bioorthogonal Metabolic Labeling Utilizing Protein Biosynthesis for Dynamic Visualization of Nonenveloped Enterovirus 71 Infection

Bioorthogonal metabolic labeling through the endogenous cellular metabolic pathways (e.g., phospholipid and sugar) is a promising approach for effectively labeling live viruses. However, it remains a big challenge to label nonenveloped viruses due to lack of host-derived envelopes. Herein, a novel bioorthogonal labeling strategy is developed utilizing protein synthesis pathway to label and trace nonenveloped viruses. The results show that l -azidohomoalanine (Aha), an azido derivative of methionine, is more effective than azido sugars to introduce azido motifs into viral capsid proteins by substituting methionine residues during viral protein biosynthesis and assembly. The azide-modified EV71 (N 3 -EV71) particles are then effectively labeled with dibenzocyclooctyl (DBCO)-functionalized fluorescence probes through an in situ bioorthogonal reaction with well-preserved viral infectivity. Dual-labeled imaging clearly clarifies that EV71 virions primarily bind to scavenger receptors and are internalized through clathrin-mediated endocytosis. The viral particles are then transported into early and late endosomes where viral RNA is released in a low-pH dependent manner at about 70 min postinfection. These results first reveal viral trafficking and uncoating mechanisms, which may shed light on the pathogenesis of EV71 infection and contribute to antiviral drug discovery.