A toolbox to visualize vRNA, viral proteins, and host factors.
We set out to develop a toolbox to visualize vRNA, viral proteins, host factors and their interactions. Previously, we produced viruses containing SunTag epitopes, allowing us to visualize translation of the viral genomic RNA (described in detail in Nature Protocols, 2025, in press). In the current project, we set out to produce viruses containing an RNA aptamer, a structured RNA element that upon binding of an otherwise non-fluorescent dye becomes fluorescent. Our first system allowed us to visualize viral RNA in living cells in the mid- and late-phases of infection, but appeared too insensitive to visualize the incoming viral RNA. To also detect the incoming viral genomic RNA, we are now employing a second RNA aptamer system. This system appears more sensitive and allows us to detect the incoming viral RNA at the single molecule level. Moreover, we set out to develop systems to visualize viral proteins during infection in living cells. For this, we use a split GFP system in which a small part of the GFP molecule is genetically fused to the viral protein of interest. Using cells expressing the remainder of the GFP molecule, we managed to construct a virus that allows us to visualize the synthesis, localization, and dynamics of the viral 2C protein in living cells. In addition, we managed to construct a two-color split-GFP virus allowing us to study two different fluorescent viral proteins at the same time.
Visualize and study viral RNA translation, replication and assembly.
To visualize and study where viral RNA replication and assembly takes place and where the viral RNA, viral proteins and host factors localize and interact in living cells, we have constructed viruses containing (combinations of) SunTag, RNA aptamers, and split-GFP viral proteins. In parallel, we implemented a novel fixed cell imaging method (4i) in our lab to dissect the composition of the sites of vRNA translation, replication and encapsidation in unprecedented detail. To study the molecular composition and organization of the viral replication complexes (i.e. the ensemble of the viral proteins that replicate the viral RNA) and the virus-induced replication organelles with which these replication complexes are associated, we have set up a cryo-electron tomography (cryo-ET) pipeline. We are using split-GFP labeled viral replication proteins and correlative light and EM microscopy (CLEM) in combination with cryo-ET to identify the viral replication and assembly complexes.
