Fig 2: eRF3a degraders synergize with G418 to rescue G542X-CFTR function.(A–D) TECC-24 measurements of the UNCCF13T cell line (G542X/G542X) treated with 0.1% DMSO or combinations of 200 μM G418, 0.1 μM CC-90009, 0.2 μM SJ6986, 0.3 μM Smg1i, and 3 μM VX-809. (A and B) Change in Ieq in response to FSK (A) and CFTRinh-172 (B). (C and D) TECC-24 tracings representing 4 replicates. All data were analyzed using ordinary linear models and are presented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: The eRF3a degrader CC-90009 rescues W1282X-CFTR in a panel of cell lines and parent primary cells.(A–C) TECC-24 measurements of the UNCX2T cell line (W1282X/W1282X) treated with 0.1% DMSO, 200 μM G418, 0.3 μM SMG1i, 0.1 μM CC-90009, and 3 μM VX-809, alone or in combination as indicated. Acute addition of the potentiator 10 μM VX-770 is indicated by an arrow. (A) TECC-24 tracing representing 3–4 replicates. (B and C) ΔIeq in response to FSK (B) and CFTRinh-172 (C). Data were analyzed using ordinary linear models. n = 3–4. (D–I) TECC-24 measurements of a panel of W1282X/W1282X cell lines and parent cells treated with 0.1% DMSO or 0.1 μM CC-90009. (D and E) TECC-24 tracing of the UNCX2T cell line (D) and parent cells (E). Tracings are representative of the W1282X/W1282X panel containing 3 cell donors with 6 replicates per donor. (F–I) ΔIeq in response to FSK (F) and CFTRinh-172 (G), benazmil (H), and basal Ieq (I). Data were analyzed using a linear mixed-effects model with the donor as a random effect factor. n = 6 per donor. Post hoc comparisons were performed using the general linear hypothesis test. All data are presented as the mean ± SD. ***P < 0.001.

Fig 4: GSPT1 associates with EBOV polymerase(A–C) Shown are anti-FLAG pull-downs of GSPT1 complexes.(A) Coimmunoprecipitation of FLAG-GSPT1 with EBOV_pol complex (L-VP35), with or without VP30.Immunoprecipitation of FLAG-GSPT1 with (B) VP35 or (C) VP30 expression alone.Representative results from two biological replicates are shown. Each blot was sliced into several parts to probe for different target proteins and imaged separately.(D) Confocal immunofluorescent analysis of GSPT1 and EBOV_pol (by VP35-HA) localization in the context of the EBOV MG system in HEK293T cells. The bottom panel shows the subcellular distribution of GSPT1 alone as a negative control. Representative images from two biological replicates are shown. Scale bars, 10 μm.See also Figure S3.

Fig 5: Effect of siRNA targeting high-confidence EBOV_pol interactors on viral infection(A) siRNA screen with authentic EBOV infection.(B) Normalized values displayed in heat maps of the relative (Rel.) percentage of EBOV infection and relative cell count. Each value is the mean of technical triplicates transfected with the same siRNA. Multiple unpaired t tests were performed to determine the statistical significance of siRNA-mediated changes on percent EBOV infection compared with that of NSC. The determined p values were log transformed, and those with p > 0.05 were excluded.(C) Individual genes for which siRNAs significantly affected infection are shown as data points in a bubble plot; data point colors and sizes correspond to the siRNA tested and log-transformed p value, respectively. Data points with multiple siRNAs significantly modulating EBOV infection are considered true hits and are outlined in white, with the corresponding gene also labeled in white. One label of each true hit is shown in red for display purposes. NPC1 used as a positive control is labeled in yellow. GSPT1 is labeled in black.(D) Representative images of selected Huh7 monolayers at 48 h post-infection (hpi). One representative result from two rounds of siRNA screen using different multiplicities of infection (MOIs) (PFUs/cell) is shown. Scale bars, 100 μm.See also Figure S2 and Table S2.