Bioorthogonal metabolic labeling utilizing protein biosynthesis for dynamic visualization of non-enveloped enterovirus 71 infection.

Bioorthogonal metabolic labeling through 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 non-enveloped viruses due to lack of host-derived envelopes. Herein, a novel bioorthogonal labeling strategy is developed utilizing protein synthesis pathway to label and trace non-enveloped 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 azido-modified EV71 (N3-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 minutes post-infection. These results firstly reveal viral trafficking and uncoating mechanisms, which may shed a light on the pathogenesis of EV71 infection and contribute to anti-viral drug discovery.