Abstract
Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we used a microscopy-based approach to image CHIKV nsP3 inside human cells. The SNAP system consists of a self-labelling enzyme tag, which catalyses the covalent linking of exogenously supplemented synthetic ligands. Genetic insertion of this tag resulted in viable replicons and specific labelling while preserving the effect of nsP3 on stress granule responses and co-localisation with GTPase Activating Protein (SH3 domain) Binding Proteins (G3BPs). With sub-diffraction, three-dimensional, optical imaging, we visualised nsP3-positive structures with variable density and morphology, including high-density rod-like structures, large spherical granules, and small, low-density structures. Next, we confirmed the utility of the SNAP-tag for studying protein turnover by pulse-chase labelling. We also revealed an association of nsP3 with cellular lipid droplets and examined the spatial relationships between nsP3 and the non-structural protein 1 (nsP1). Together, our study provides a sensitive, specific, and versatile system for fundamental research into the individual functions of a viral non-structural protein during infection with a medically important arthropod-borne virus (arbovirus).
Highlights
The 11.8-kilobase, single-stranded, positive-sense-RNA genome of Chikungunya virus (CHIKV) encodes four non-structural proteins and three main structural proteins
We expand the panel of fluorescent CHIKV derivatives by generating a replicon that encodes SNAP-tagged non-structural protein 3 (nsP3)
By developing protocols for the visualisation of SNAP-nsP3 that are compatible with recently developed imaging technologies such as Airyscan super-resolution confocal imaging, we lay the foundation for more detailed studies of nsP3 function within the cellular host environment
Summary
The 11.8-kilobase, single-stranded, positive-sense-RNA genome of CHIKV encodes four non-structural proteins (nsP1, nsP2, nsP3, nsP4) and three main structural proteins (capsid and envelope glycoproteins E1/E2). Specific sequence motifs within the C-terminal domain are required to recruit cellular host factors such as Amphiphysin-1 and -2 (Amph[1] and 2) and the GTPase Activating Protein (SH3 Domain) Binding Protein (G3BP)[27, 29,30,31,32,33,34,35,36,37,38,39] This domain can accept insertion of foreign proteins such as luciferase, green fluorescent protein (GFP), and mCherry[26, 30, 31, 40,41,42,43]. Efforts to develop anti-CHIKV treatments will benefit from an improved understanding of CHIKV biology, including a better grasp of the various roles of nsP3 that antiviral strategies could target in the future
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