Live-cell imaging of a plant virus replicase during infection using a genetically encoded, antibody-based probe

Replication of plant positive-strand RNA viruses occurs in association with intracellular membranes. To date, no versatile technology has been developed to directly label and visualize an active replicase in live plant cells because, in general, replicase function is not retained when it is fused to a protein for fluorescence imaging. We developed a technique to label and image a plant virus replicase during infection using the transiently expressed human influenza hemagglutinin (HA) frankenbody (FB), an antibody fragment that binds the HA epitope. The function of FB was demonstrated by visualizing the targeting of mCherry-fused FB (FB-mCherry) to an HA-tagged and GFP-fused endoplasmic reticulum (ER) marker protein in its native location. The combination of a two-component inducible system with the FB probe enabled FB-mCherry to label an HA epitope-tagged, functional replicase of Plantago asiatica mosaic virus (PlAMV) during its infection in Nicotiana benthamiana cells without affecting virus replication efficiency. The HA-tagged PlAMV replicase forms punctate structures associated with the ER and plasmodesmata and is localized in the vicinity of dsRNA, a hallmark of a viral replication complex. This application of epitope tag-binding intracellular probes for live subcellular imaging in plant cells offers insights into the localization dynamics of an active plant virus replicase during infection and the potential to co-localize host factors with the active replicase in situ.