Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time

Abstract

The influenza virus uses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging. Enveloped viruses such as the influenza virus cause significant disease in humans. In order to cause a productive infection, the virus particle must interact with the host cell using the viral proteins encoded within its genome. For many such viruses, it is possible to directly observe the early steps in infection, yet for technical reasons it has been extremely difficult to study the genesis of daughter virions as they bud off of infected host cells. Here we devised a chemoenzymatic labeling strategy to site-specifically append probes to the influenza hemagglutinin (HA) and neuraminidase (NA) proteins using the bacterial sortase A enzyme. Because labeling is confined to surface exposed HA and NA in the context of live, infected cells, it is possible to study budding biochemically and microscopically in real-time. Using this system, we can observe budding of flu virions from discrete sites at the cell surface. Our work will enable detailed investigation into the birth of viruses from infected host cells and can likely be applied to viruses other than influenza that have been similarly resistant to real-time microscopic observation during budding.