Abstract
Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDVE2_fluo). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDVE2_fluo particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDVE2_fluo as a tool to study the dynamics of the whole life cycle of BVDV in real time.
Highlights
Bovine viral diarrhoea virus (BVDV) is an economically important pathogen within the family Flaviviridae, genus Pestivirus and has been extensively used as a model for hepatitis C virus research
The attachment and entry of BVDV or any other pestivirus has never been analysed in real time as suitable labelled viruses have been lacking to date
To gain dynamic information of virus entry, viruses can be genetically labelled by the fusion of a structural protein with a fluorophore or by labelling components of intact virus particles with a fluorescent dye
Summary
Bovine viral diarrhoea virus (BVDV) is an economically important pathogen within the family Flaviviridae, genus Pestivirus and has been extensively used as a model for hepatitis C virus research. BVDV particles consist of a nucleocapsid, composed of core protein and the viral genome, and a viral envelope, in which three surface glycoproteins, Erns, E1 and E2, are anchored. To gain dynamic information of virus entry, viruses can be genetically labelled by the fusion of a structural protein with a fluorophore or by labelling components of intact virus particles with a fluorescent dye (e.g. adenovirus/enveloped particles with DiD) (reviewed in[7]). Within the Flaviviridae, infection dynamics have been analysed for DiD labelled Dengue virus, revealing diffusion patterns on the cell surface and the recruitment dynamics of endosomal components during virus entry[8]. In the present paper we describe the generation and characterisation of BVDV particles that are labelled with a fluorophore fused to E2 surface glycoprotein and their applicability for the imaging of infection dynamics
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