Fig 2: L1TD1 affects the dynamics of SG formation in H1 hESCs. Forty-eight hours after siRNA transfection, control (A) and L1TD1-depleted (B) cells were treated with 75 μM arsenite for the indicated times. The proportions of cells without discrete G3BP1 granules at 30 min were 16.23 ± 1.93% (A) and 4.93 ± 0.69% (B), respectively (P = 0.0007).

Fig 3: An SFHV infection induces activation of the SG response pathway and redistribution of G3BP1 in SHFV-infected cells. (A–D) MA104 cells were mock-infected or infected with SHFV at the indicated MOI. Uninfected cells were treated with SA (0.5 mM) for 30 min at 37°C. Whole-cell lysates were harvested in 1× RIPA buffer at the indicated times and used for WB analysis with antibodies specific for (A) PKR, p-PKR, SHFV nsp1β or (B, D) eIF2α, p-eIF2α, or SHFV nsp1β. (C) Relative intensity of p-eIF2α protein bands in mock-infected, SA-treated or SHFV-infected cells. WB band intensity was quantified using ImageJ software and normalized to total eIF2α. Values are ±SEM and significance was analyzed using an unpaired Student’s t-test; *P ≤ 0.05. The data shown are representative of three independent experiments. (E) MA104 cells were mock-infected or infected with SHFV (MOI of 0.1). Uninfected MA104 cells were treated with 0.5 mM of SA for 30 min at 37°C. At the indicated times, cells were fixed, permeabilized, and incubated with anti-G3BP1 antibody (green) and anti-dsRNA antibody (red). Nuclei were stained with Hoechst 33342 (blue). (F) Mock-infected images and enlarged portions of the SHFV 24 hpi images shown in panel E. Fluorescence intensity profiles of the G3BP1 (green) and dsRNA (red) fluorescent signals along three different colored cross-section lines in the enlarged merged image of infected MA104 cells were generated with ImageJ software. (G) Primary macaque MΦs were mock-infected or infected with SHFV (MOI of 0.1). The cells were fixed and processed for IFA at 24 hpi. Fluorescence intensity profiles of the G3BP1 (green) and dsRNA (red) fluorescent signals along three different colored cross-section lines of an enlarged merged IFA image are shown. In this and subsequent figures, images were obtained with a Zeiss Axio Observer Z1 fluorescent microscope and a 40× objective. Scale bars, 100 µm. Enlarged images scale bars, 50 µm.

Fig 4: The SHFV nsp2 and N proteins colocalize with G3BP1 foci in infected or transfected cells. (A) MA104 cells were mock-transfected or transfected with mRNA encoding nsp1α or nsp1β with a C-terminal V5 tag for 20 h or transfected with plasmid DNA encoding nsp1γ, nsp2, nsp3, or N with a C-terminal V5 tag for 48 h. Cells were fixed, permeabilized, and incubated with antibodies specific for G3BP1 (green) and V5 (red). Nuclei were stained with Hoechst 33342 (blue). (B) Enlargement of a single nsp2-transfected cell from panel (A). Fluorescence intensity profiles of G3BP1 (green) and V5 (red) signals along the yellow cross-section line in the enlarged merged image are shown. (C) Enlargement of N-transfected cells from panel (A). Fluorescence intensity profiles of G3BP1 (green) and V5 (red) signals along the yellow cross-section line in the enlarged merged image are shown. (D and E) MA04 cells were infected with SHFV (MOI of 0.1) for 4 h and then mock-transfected or transfected with nsp2 or N plasmid DNA for 20 h. At 24 hpi, the cells were fixed, permeabilized, and incubated with antibodies specific for G3BP1 or dsRNA (green) and V5 (red). Nuclei were stained with Hoechst 33342 (blue). Fluorescence intensity profiles for G3BP1 or dsRNA (green) and V5 (red) signals along the yellow cross-section lines in the enlarged merged images are shown. (F) MA104 cells were mock-transfected or transfected with mRNA encoding nsp1α or nsp1β with a C-terminal V5 tag or transfected with plasmid DNA encoding nsp2, nsp3, or N with a C-terminal V5 tag. At 20 hpt, transfected cells were treated with 2 mM SA or 2 mM DTT for 45 min. Cells were fixed, permeabilized, and incubated with antibodies specific for G3BP1 (green) and V5 (red). Nuclei were stained with Hoechst 33342 (blue).

Fig 5: G3BP1 interacts with the SHFV nsp2 and nucleocapsid proteins but not with nsp1α, nsp1β, nsp1γ, or nsp3. (A) Whole-cell lysates were harvested in IP lysis buffer at 24 hpi from mock-infected or SHFV-infected (MOI of 0.1) MA104 cells. G3BP1 was immunoprecipitated using rabbit anti-G3BP1 antibodies. A rabbit anti-IgG antibody was used as an antibody control. Immunoprecipitated proteins were analyzed by WB using mouse anti-G3BP1 antibodies or rabbit anti-nsp1β or anti-nsp1γ antibodies. (B and C) MA104 cells were mock-transfected or transfected with mRNA encoding nsp1α, nsp1β or nsp1γ, each with a C-terminal V5 tag. (B) At 20 h post transfection (hpt), whole-cell lysates were harvested in 1× RIPA buffer and used for WB analysis with antibodies specific for V5 or nsp1γ. (C) At 20 hpt, whole-cell lysates were harvested in IP lysis buffer and the V5-tagged nsp1 proteins were immunoprecipitated using V5-trap magnetic agarose beads. Immunoprecipitated proteins were analyzed by WB using anti-G3BP1 and anti-V5 antibodies. (D–F) MA104 cells were mock-infected or infected with SHFV (MOI of 0.1) for 4 h, then independently transfected with mRNA encoding (D) nsp1α, (E) nsp1β, or (F) nsp1γ for 20 h. At 24 hpi, whole-cell lysates were immunoprecipitated using V5-trap magnetic agarose beads. Immunoprecipitated proteins were analyzed by WB using anti-G3BP1 and anti-V5 antibodies. (G–I) MA104 cells were mock-transfected or transfected with plasmid DNA encoding (G) nsp2, (H) nsp3, or (I) N protein for 48 h and then mock-infected or infected with SHFV (MOI of 0.1) for an additional 24 h. At 72 hpt, whole-cell lysates were immunoprecipitated with rabbit anti-G3BP1 antibody. Immunoprecipitated proteins were analyzed by WB using mouse anti-G3BP1 and mouse anti-V5 antibodies. (J) Whole-cell lysates prepared from MA104 cells that were mock-transfected or transfected with nsp2, nsp3, or N protein plasmid DNA for 72 h were immunoprecipitated using V5-trap magnetic agarose beads. Immunoprecipitated proteins were analyzed by WB using antibodies specific for G3BP1 and V5.