Dynamic Tracking Of EV71 Infection Via Amino Acid Metabolism-Mediated In-Situ Bioorthogonal Labeling

Enterovirus 71(EV71)is a major agent of hand,foot and mouth disease(HFMD),which can progress to severe neurological disease such as encephalitis,meningitis,acute flaccid paralysis,cardiopulmonary failure and even death.Due in part to an insufficient understanding of the mechanism for EV71 infection,the government and society are faced with a big challenge in prevention and control of related diseases.Tracking virus infection pathway in cells is indispensable for understanding the viral infection mechanism,and the successful labeling of virions is the key to achieve virus tracking.There are several general methods for virus labeling,including genetic engineering,direct chemical coupling and viral self-assembly.However,it was difficult for spreading and applying for these methods due to several defects and limits,such as complicated operation,low labeling efficiency,limited application and inactive labeled viruses.Recently,bioorthogonal chemistry based on metabolic engineering has been widely applied to living organisms labeling and tracking due to its non-destructive,high-efficiency,and specific targeting capabilities.This strategy incorporates a special chemical reporter into target biomolecules through natural metabolism,and the probe with a complementary group is covalently linked to the chemical reporter,thereby allowing a specific labeling and tracking for target biomolecules.Considering viral components deriving from cellular metabolism,virus capsids could be effectively modified through host cell amino acid metabolism.Hence,we developed an in situ bioorthogonal labeling strategy based on the amino acid metabolism to dynamically tracking EV71 infection and dissected viral infection mechanism in depth.1.The development of viral bioorthogonal labeling based on the amino acid metabolism.L-Azidohomoalanine(Aha)was metabolically incorporated into EV71 capsid proteins during progeny virus assembly,and the modified EV71(N3-EV71)were in-situ labeled with dibenzocyclooctyl(DBCO)-functionalized fluorescence probes through bioorthogonal reactions after adhering to host cell surface.The results indicated N3-EV71 could be rapidly and efficiently labeled by DBCO-probes in host cells with effectively preserving viral infectivity,which provided a reliable technique tools for future virus tracking.2.Dynamical tracking of EV71 infection using the in situ bioorthogonal labeling strategy.We separately labeled the viral capsid and nucleic acid by in situ labeling and SYTO RNA stain to achieve a dual-labeling for EV71.The results showed EV71 binding to scavenger receptors on cell surface quickly entered into host cells by clathrin-mediated endocytosis,and then transported into late endosomes for RNA release.These data elucidated clearly EV71 infection route and the mechanism of RNA uncoating in the early stagy.In this study,we proposed and developed the in situ bioorthogonal labeling strategy based on amino acid metabolism for dynamically tracking viral infection,and clearly dissected the infection mechanism of EV71 in the early stage,which could assist in the development of anti-EV71 therapies.