The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

1. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   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.
2. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   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.
3. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   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.
4. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   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.

• Copyright © 2014, American Society for Microbiology. All Rights Reserved.