The C-terminal repeat domains of nsP3 from the Old World alphaviruses bind directly to G3BP

Abstract

The Old World alphaviruses block stress granule assembly by sequestration of RasGAP SH3-domain binding protein (G3BP). Here, we show that the proline-rich sequences in the hypervariable domain of nonstructural protein 3 (nsP3) of both Semliki Forest virus and Chikungunya virus were dispensable for binding to G3BP. nsP3 variants with or without this domain colocalized with G3BP. Furthermore, we show that the C-terminal repeat motifs of nsP3 were sufficient for G3BP binding.

FIG 1

SFV and CHIKV nsP3 sequences binding the SH3 domain of amphiphysins are not required for G3BP-1 binding. (A) MEFs were infected with SFV wt or SFV-ΔP1+2 at an MOI of 1. At 8 hpi, cells were fixed and stained for nsP3 and G3BP-1. Results are representative of two independent experiments. Images of single focal planes were processed using Adobe Photoshop. Bar, 10 μm. (B) Extreme C-terminal sequences of SFV wt nsP3, nsP3-ΔP1+2, nsP3-ΔC, and CHIKV wt nsP3 and nsP3-ΔP1. The C-terminal repeat sequences are in bold type. (C) BHK cells were mock transfected or transfected with the indicated constructs. Cell lysates were prepared 16 h after transfection, precipitated with streptavidin-coated beads, and separated by SDS-PAGE. Lysates and precipitates were probed with streptavidin and with G3BP-1 and actin antisera. Results are representative of three independent experiments. IP, immunoprecipitation.

FIG 2

SFV and CHIKV nsP3 sequences binding the SH3 domain of amphiphysins are not required for localization to G3BP-1-positive foci. MEFs were mock transfected or transfected with pEBB/PP-SFVnsP3, nsP3-ΔP1+2, or nsP3-ΔC or pEBB/PP-CHIKVnsP3 or nsP3-ΔP1. After 22 h, transfected cells were mock treated (A) or treated with sodium arsenite (B) for 1 h, fixed and stained with streptavidin (for nsP3) and with G3BP-1 and TIA-1 antisera, and analyzed by confocal microscopy. Images of single focal planes were processed using Adobe Photoshop. Bars, 10 μm. Profiles were calculated using the RGB profiler tool in ImageJ.

FIG 3

CHIKV nsP3 sequences binding the SH3 domain of amphiphysins are not required for localization to G3BP-1-positive foci in Vero cells. (A) Amino acid sequence of Δ398–406 mutation. (B) Vero cells were mock transfected or transfected with pEBB/PP vector or with CHIKVnsP3 or nsP3-Δ398–406. Cell lysates were prepared 22 h after transfection, precipitated with streptavidin-coated beads, and separated by SDS-PAGE. Lysates and precipitates were probed with streptavidin and with G3BP-1 and actin antisera. Results are representative of three independent experiments. (C) Vero cells were mock transfected or transfected with pEBB/PP-CHIKVnsP3 or nsP3-Δ398–406. At 22 h, transfected cells were mock treated (left panels) or treated with sodium arsenite (right panels) for 1 h, fixed and stained with Cy3-streptavidin (for nsP3) and with G3BP-1 and TIA-1 antisera, and analyzed by confocal microscopy. Images of single focal planes were processed using Adobe Photoshop. Bar, 10 μm. Profiles were calculated using the RGB profiler tool in ImageJ.

FIG 4

The C-terminal repeat motifs of SFV and CHIKV nsP3 are sufficient for G3BP binding. (A) Amino acid sequences of the C termini of the SFV-31 and CHIKV-51 constructs used in this study. The repeat sequences are underlined. (B) BHK cells were mock transfected or transfected with pEGFP-SFV-31, pEGFP-CHIKV-51, or pEGFP-C1. Cell lysates were prepared 16 h after transfection, immunoprecipitated with GFP antisera, and separated by SDS-PAGE. Lysates and precipitates were probed for G3BP-1, G3BP-2, GFP, and actin antisera. Results are representative of three independent experiments. (C) Purified GST, GST-nsP3-31, or His-G3BP-1-NTF2 protein was mixed, immunoprecipitated with GST antisera, and separated by SDS-PAGE. Lysates and precipitates were probed with GST or His antisera. Data are representative of three independent experiments.