Single-Virus Microscopy of Biochemical Events in Viral Entry

Cell entry by enveloped viruses involves a set of multistep, multivalent interactions between viral and host proteins as well as manipulation of nanoscale membrane mechanics by these interacting partners. A mechanistic understanding of these events has been challenging due to the complex nature of the interactions and the event-to-event heterogeneity involved. Single-virus microscopy has emerged as a powerful technique to probe viral binding and fusion kinetics. Single-event distributions compiled from individual viral particle measurements have enabled estimates of protein stoichiometry at fusion interfaces, a better understanding of the rate-limiting steps for fusion, and a more robust identification of the biochemical regulatory factors for viral entry. Recent technical advances have made these experiments feasible on less specialized microscopes, increasing their accessibility to a broad range of scientists. Single-virus entry kinetics have now been measured for a wide range of enveloped viruses and on both synthetic and physiological substrates. Here, we briefly review the major progress in the area. We then describe the critical apparatus, protocols, analytical techniques, and optimizations needed for robust measurements of virus-membrane interactions.