A Tale of 20 Alphaviruses; Inter-species Diversity and Conserved Interactions Between Viral Non-structural Protein 3 and Stress Granule Proteins

Abstract

Alphaviruses infect a diverse range of host organisms including mosquitoes, mammals, and birds. The enigmatic alphavirus non-structural protein 3 (nsP3) has an intrinsically disordered, C-terminal hypervariable domain (HVD) that can interact with a variety of host proteins associated with stress granules (SGs). The HVD displays the highest variability across the more than 30 known alphaviruses, yet it also contains several motifs that are conserved amongst different subgroups of alphaviruses. For some alphaviruses, specific nsP3-SG protein interactions are essential for virus replication. However, it remains difficult to attribute general roles to these virus-host interactions, as multiple amino acid motifs in the HDV display a degree of redundancy and previous studies were performed with a limited number of alphaviruses. To better understand nsP3-host protein interactions we conducted comprehensive co-localization experiments with the nsP3s of 20 diverse alphaviruses: chikungunya, Semliki Forest, Sindbis, Bebaru, Barmah Forest, Getah, Mayaro, Middelburg, O'nyong-nyong, Ross River QML and T48, Una, Whataroa, Southern Elephant Seal, Eilat, Tai Forest (TAFV), Venezuelan/Eastern/Western equine encephalitis (V/E/WEEV) and the aquatic Salmonid alphavirus (SAV), with three different SG proteins (G3BP and its insect homolog Rasputin, FMRP) and BIN1 in mammalian and mosquito cell lines. Despite that all terrestrial alphavirus nsP3s contained at least one BIN1-binding motif (PxPxPR), not all nsP3s co-localized with BIN1. Further, all alphaviruses except SAV, TAFV and VEEV displayed co-localization with G3BP. Although viruses lacking FGxF-like motifs contained Agenet-like domain binding motifs to facilitate interaction with FMRP, cytoplasmic nsP3 granules of all tested alphaviruses co-localized with FMRP. Crispr-Cas9 knockout of G3BP in mammalian cells abolished nsP3-FMRP co-localization for all alphaviruses except V/E/WEEV nsP3s that bind FMRP directly. G3BP knockout also changed nsP3 subcellular localization of Bebaru, Barmah Forest, Getah, and Sindbis viruses. Taken together this study paints a more detailed picture of the diverse interactions between alphavirus nsP3 and SG-associated host proteins. The interaction between nsP3 and G3BP clearly plays a central role and results in recruitment of additional host proteins such as FMRP. However, direct binding of FMRP can make the interaction with G3BP redundant which exemplifies the alternate evolutionary paths of alphavirus subgroups.

Keywords: BIN1; FMRP; G3BP; alphavirus; hypervariable domain; nsP3; stress granules.

Figure 1

Evolutionary relationships between alphavirus nsP3 sequences. A Bayesian phylogeny of alphavirus nsp3 coding sequences is shown a GTR + I + G model. Bayesian posterior probabilities and maximum likelihood values (100 replicates) are shown before and after the slash respectively. The scale bar represent 0.02 expected substitutions per site. A dash (-) indicates values <50.

Figure 2

Part of an alignment of 20 alphavirus nsP3 HVDs based on three host protein-binding motifs. In blue; the proline-rich BIN1-binding motif, PxPxPR. In blue and bold; high affinity proline-rich motifs, P[I/V][P/A]PPR[R/K/P][R/K][R/K]. In yellow; Agenet-like domein binding motif which interacts with FMRP. In green; G3BP-binding motifs, the G3BP non-conicial motif (CHIKV), the LITF motif (EEEV), and the FGDF motifs. In green and bold, the fourth residue of the FGDF motifs is either an isoleucine or leucine. (*) part of the alignment not shown.

Figure 3

Alphavirus nsP3 co-localization with G3BP. HeLa cells were transfected with alphavirus nsP3-eGFP, stained with antibodies for G3BP and visualized by immunofluorescence. Green indicates localization of nsP3, red represents G3BP. NsP3-G3BP co-localization is visualized in yellow in the overlay. The percentages in the overlay images represent co-localization percentage of all counted cells. A distinction is made for alphaviruses with one or two FGDF-like motifs or the presence of one or two Agenet-like domain binding motifs.

Figure 4

NsP3-G3BP co-localization percentages. HeLa cells with co-localization of nsP3 and G3BP are represented as co-localization percentages. Bars represent the mean co-localization pattern of 30–50 transfected HeLa cells. A distinction is made for alphaviruses with zero, one, or two FGDF-like motifs. In absence of cytoplasmic granules, no co-localization could be determined, these nsP3-eGFPs are indicated with (+).

Figure 5

Alphavirus nsP3 co-localization with RIN in mosquito cells. C6/36 cells were transfected with alphavirus nsP3-eGFP and pPUB-RIN. Green indicates localization of nsP3, red represents RIN. NsP3-RIN co-localization is visualized in yellow in the overlay. The percentages in the overlay images represent co-localization percentage of all counted cells. A distinction is made for alphaviruses with zero, one, or two FGDF-like motifs.

Figure 6

NsP3-RIN co-localization percentages in mosquito cells. Cells with co-localization of nsP3 and RIN are represented as co-localization percentages. Bars represent the mean co-localization pattern of 50–100 transfected C6/36 cells. A distinction is made for alphaviruses with zero, one, or two FGDF-like motifs.

Figure 7

Alphavirus nsP3 co-localization with FMRP in wildtype and G3BP KO cells. HeLa wt and HeLa G3BP KO cells were transfected with alphavirus nsP3-eGFP, stained with antibodies for FMRP and visualized by immunofluorescence. Green indicates localization of nsP3, red represents FMRP. NsP3-FMRP co-localization is visualized in yellow in the overlay. The percentages in the overlay images represent co-localization percentage of all counted cells. A distinction is made for alphaviruses with one or two FGDF-like motifs or the presence of one or two Agenet-like domain binding motifs.

Figure 8

Alphavirus nsP3 co-localization with FMRP in wildtype and G3BP KO cells. HeLa wt and HeLa G3BP KO cells were transfected with alphavirus nsP3-eGFP, stained with antibodies for FMRP and visualized by immunofluorescence. Green indicates localization of nsP3, red represents FMRP. NsP3-FMRP co-localization is visualized in yellow in the overlay. The percentages in the overlay images represent co-localization percentage of all counted cells. A distinction is made for alphaviruses with one or two FGDF-like motifs or the presence of one or two Agenet-like domain binding motifs.

Figure 9

NsP3-FMRP co-localization percentages in wildtype and G3BP KO cells. Cells with co-localization of nsP3 and FMRP are represented as co-localization percentages. Bars represent the mean co-localization pattern of 50-100 transfected HeLa wt and HeLa G3BP KO cells. A distinction is made for alphaviruses with one or two FGDF-like motifs or the presence of one or two Agenet-like domain binding motifs. In absence of cytoplasmic granules, no co-localization could be determined, these nsP3-eGFPs are indicated with (+).