Site-Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π-Clamp-Mediated Cysteine Conjugation.

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells.