Single-Virus Microscopy\nof Biochemical Events in Viral\nEntry

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