SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy

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).

Alphavirus nsP3 ADP-ribosylhydrolase Activity Disrupts Stress Granule Formation

“…In cells transfected with full-length nsP3, nsP3 co-localized with G3BP1, but not with eIF3b in the majority of cells examined (73%; Fig. 1a, c), as previously reported [36,42]. In contrast, in cells transfected with the nsP3 HVD alone, the nsP3 HVD co-localized with both SG components eIF3b and G3BP1 in 93% of cells examined (Fig.…”

“…S1c). Taken together with work from others [36,[40][41][42], these data suggest that expression of full-length nsP3 alters the association of individual SG components (e.g., release of eIF3b and retention of G3BP1 with nsP3), and it is likely that domain(s) other than HVD of nsP3 may be responsible for suppressing SG formation.…”

“…Overexpression of alphaviral nsP3 alone suppresses the formation of SGs even in the presence of the classic SG inducer arsenite [36,[40][41][42]. The current paradigm is that the essential SG component G3BP1 is sequestered by nsP3 in another class of cytoplasmic structure that does not contain the Page 6 canonical SG marker translation initiation factor eIF3b ( Fig.…”

“…For example, the mosquito-borne alphaviruses, which cause a range of diseases from rashes and arthritis to encephalitis, induce SG formation transiently in the early stage of infection, followed by disassembly [32,[34][35][36]. Previous studies have identified the alphaviral nonstructural protein 3 (nsP3), a key factor for virus replication and virulence [37][38][39], as able to suppress SG assembly [36,[40][41][42]. nsP3 is a tripartite protein composed of a highly conserved…”

The Interplay of Viral and Host Factors in Chikungunya Virus Infection: Targets for Antiviral Strategies

“…Instead a significant reduction in total infectious viral titer was observed in Aedes albopictus in vivo [195]. Interestingly, Remenyi and colleagues also showed that nsP3 is closely associated with the host cellular lipid droplets and also with nsP1 [196].…”