Virus Labeling Via Biological Metabolism And Bioorthogonal Chemistry

Viral diseases are a great threat to human health.Real-time tracking of the dynamic process of virus invasion and understanding the invasion mechanisms of virus infection is crucial to prevent and treat viral diseases.For successful tracking,efficient labeling methods are indispensable.The key is to effectively connect the tags to virus components,and do not affect the structure and function of the virus.The current methods failed to solve this problem well.In this paper,both biosynthesis and metabolic incorporation were simultaneously utilized to introduce functionalized groups to the virus.Such functionalized groups could be fluorescently labeled via a bioorthogonal reaction.These labeling methods were used to real-time tracking of the dynamic process of vaccinia virus（VACV）invasion,which is of importance to correctly understand invasion mechanisms of VACV.The three main contents are as follows:1.Enveloped virus labeling via both biosynthesis and metabolic incorporation of phospholipids in host cellsThe choline（Cho）analogue azide-choline（azide-Cho）was biosynthetically bonded to phospholipids and incorporated into the host cells,and subsequently virus was propagated,then the progeny virions with the azide-modified envelope formed from the membranes of the 400μm azide-modified host cells were harvested.The virions could be labeled with fluorophores conveniently through strain promoted azide-alkyne cycloaddition（SPAAC）with a high efficiency and good biocompatibility.A‘molecular light switch’complex[Ru（phen）₂（dppz）]²⁺has been exploringly used to label vaccinia virus nucleic acid with the“virus replication-intercalated labeling”.By combining the two,the living virus was also simultaneously dual-labeled in host cells.The labeling efficiency is above 85%,and this method can be applied to all DNA or RNA enveloped viruses with complex secondary structures.The labeled viruses were fully infectious,and can be applied to single particle tracking.2.Integrating two efficient and specific bioorthogonal ligation reactions with natural metabolic incorporation in one cell for virus dual labelingTaking advantage of bioorthogonal reaction characteristics with small size and high specificity reaction groups,we synthesised methionine analogues---azidohomoalanine（AHA）and deoxythymidine analogues---5-Vinyl-2’-deoxyuridine（VdU）,respectively.Add 400μm AHA and 40μm VdU after virus replication.AHA and VdU were incorporated into viral protein and nucleic acids by their intrinsic biosynthetic machinery,and then the virus was dual-labeled via SPAAC and inverse electron-demand Diels-Alder reaction（IEDDA）.Both envelope virus and capsid virus could be labeled with a high efficiency,the labeling efficiency more than 83%.The labeled viruses were structurally intact and fully infectious.This method was more universality,it can be used for labeling both enveloped virus and capsid virus.3.The study of invasion mechanism of VACVThe invasion process of the labeled viruses was studied by fluorescent imaging system.We combined with other cell biology methods,such as fluorescent colocalized analysis,fluid uptake,drug inhibition experiment,etc.It was found that the VACV entered cells through macropinocytosis mechanism,which was actin-dependent but clathrin-,caveolin-independent endocytosis that leads to internalization of fluid and membrane into large vacuoles.These results would promote the progress of explaining VACV invasion process and mechanism,and provid important basis to correctly understand VACV pathogenic mechanism.