Elucidating virus entry using a tetracysteine-tagged virus.

Fluorescent tags constitute an invaluable tool in facilitating a deeper understanding of the mechanistic processes governing virus-host interactions. However, when selecting a fluorescent tag for in vivo imaging of cells, a number of parameters and aspects must be considered. These include whether the tag may affect and interfere with protein conformation or localization, cell toxicity, spectral overlap, photo-stability and background. Cumulatively, these constitute challenges to be overcome. Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a non-enveloped virus that is comprised of two architecturally complex capsids. The outer capsid, composed of two proteins, VP2 and VP5, together facilitate BTV attachment, entry and the delivery of the transcriptionally active core in the cell cytoplasm. Previously, the significance of the endocytic pathway for BTV entry was reported, although a detailed analysis of the role of each protein during virus trafficking remained elusive due to the unavailability of a tagged virus. Described here is the successful modification, and validation, of a segmented genome belonging to a complex and large capsid virus to introduce tags for fluorescence visualization. The data generated from this approach highlighted the sequential dissociation of VP2 and VP5, driven by decreasing pH during the transition from early to late endosomes, and their retention therein as the virus particles progress along the endocytic pathway. Furthermore, the described tagging technology and methodology may prove transferable and allow for the labeling of other non-enveloped complex viruses.