Skip to main content
. 2021 Aug 3;10:e71047. doi: 10.7554/eLife.71047

Figure 3. OAS1 isoforms require catalytic and RNase L activity.

(A) Expression of OAS1 p42 and p46 along with their corresponding catalytic mutants (250 ng) at 24 hr post transfection in OAS1 KO 293 T cells. (B) Quantification of EMCV 5′UTR by RT-qPCR in OAS1 KO 293 T cells expressing a control EV, p42, p46, or their corresponding catalytic mutants (250 ng) at 24 hr post EMCV infection (MOI=0.001). (C) Viral titers at 24 hr post EMCV infection (MOI=0.001) taken from OAS1 KO 293 T cells transfected with a control EV, p42, p46, or their corresponding catalytic mutants. (D) Immunoblot analysis of OAS1 and RNase L at 24 hr post transfection in Cas9 or RNASEL KO 293 T cells. (E) Quantification of EMCV 5′UTR by RT-qPCR in Cas9 and RNASEL KO 293 T cells expressing a control EV, p42, or p46 at 24 hr post EMCV infection (MOI=0.001). (F) Viral titers at 24 hr post-infection with EMCV (MOI=0.001) taken from Cas9 or RNASEL KO 293 T cells transfected with control EV, p42, or p46. (G) In vitro 2′−5′A synthesis assay of OAS1 p42 and p46 isoforms and their CaaX motif mutants; a representative blot of two independently performed experiments is depicted. (H) Immunoblot analysis of OAS1 and IRF3 in WT or IRF3 KO 293FT cells at 24 hr post transfection. (I) Quantification of EMCV 5′UTR 24 hr post EMCV infection (MOI=0.001) in WT or IRF3 KO 293FT cells transfected with a control EV, p42, or p46. (B, C, E, F) and (I) each data point represents an independently performed experiment. Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (A, D) and (H) Representative immunoblots of three (A, H) and five (D) independent experiments are shown.

Figure 3—source data 1. Uncropped gels for the associated panels in Figure 3 and Figure 3—figure supplement 1.

Figure 3.

Figure 3—figure supplement 1. RNase L-dependent activity of OAS1 isoforms.

Figure 3—figure supplement 1.

(A) Expression of OAS1 p42 and p46 along with their corresponding catalytic mutants at 24 hr post transfection in OAS1 KO 293 T cells. (B) Quantification of EMCV 5′UTR by RT-qPCR from OAS1 KO 293 T cells expressing a control EV, p42, p46, or their corresponding catalytic mutants at 24 hr post EMCV infection (MOI=0.001). (C) Immunoblot analysis of RNase L expression in Cas9 and clonal RNASEL KO 293 T cells. Clone five was used for subsequent experiments. (D) Immunoblot analysis of OAS1 and RNase L expression in RNASEL KO 293 T cells at 24 hr post transfection with EV, p42, p46 with or without an RNase L expression plasmid. (E) Quantification of EMCV 5′UTR or (F) EMCV titers from RNASEL KO 293 T cells transfected as in (D) followed by infection with EMCV for 24 hr (MOI=0.001). (E) and (F) Data were analyzed using a one-way ANOVA with Dunnett’s multiple comparisons test where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (A) and (D) Representative immunoblots of (A) three and (D) five independently performed experiments are shown. (B, E) and (F) Each data point represents an independently performed experiment.