Bioorthogonal Surface Modification of Measles and Vaccinia Viruses Using Metabolic Azide Reporters

Enveloped viruses, such as measles virus (MV) and vaccinia virus (VACV), acquire their lipid bilayer envelopes from host cell membranes during viral assembly. These membranes can be chemically modified to enable selective labeling and visualization. This chapter details a metabolic labeling strategy in which Vero cells, a prototypical host cell line, are supplemented with peracetylated azido sugars (Ac₄GalNAz, Ac₄ManNAz) and azido-choline analogs (AECho). These bioorthogonal metabolic reporters are incorporated into glycan and phospholipid via biosynthetic pathways, resulting in azide-functionalized glycoconjugates and phosphatidylcholine (PC) derivatives within both host and viral membranes. The surface-exposed azido groups on progeny virions can be further conjugated with fluorescent probes or other entities via strain-promoted azide-alkyne cycloaddition (SPAAC) or copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. Fluorescent labeling enables direct visualization of viral particles by fluorescence microscopy. This chapter also provides detailed protocols for virus propagation, purification, and labeling, offering a versatile platform for live-cell imaging, viral trafficking studies, and the development of targeted delivery systems.