Fig. 5: CSpV1-dsRNAs trigger type I IFN signaling through activation of the Pkr and Rig-I/Mavs/Sting signaling pathways.

a Stable IEC4.1 cells deficient in Ifih1, Ddx58, Mavs, or Eif2ak2 generated using the CRISPR/Cas9 approach as confirmed by Western blot. Representative gel images from three independent experiments are shown. b Knockdown of Tlr3 with an siRNA in IEC4.1 cells as confirmed by RT-qPCR. Data are from three biological replicates and presented as mean values ± SD. p values were determined by two-tailed unpaired Student’s t-test. c C. parvum- and CSpV1-dsRNA-induced Ifnb1 expression in IEC4.1 cells deficient in Ifih1, Ddx58, Mavs, or Eif2ak2 and in cells treated with the Tlr3-siRNA. Cells were exposed to C. parvum or transfected with CSpV1-dsRNAs for 24 h. To knockdown Tlr3, cells were first treated with the Tlr3-siRNA for 24 h and then exposed to infection or CSpV1-dsRNA transfection. Ifnb1 expression was measured by RT-qPCR. d Inhibition of Sting signaling and Pkr signaling on C. parvum-induced expression of type I IFNs and type I IFN-controlled genes. IEC4.1 cells were exposed to C. parvum infection for 8 h and cultured for additional 16 h in the presence or absence of the inhibitor C-178 (to Sting signaling) or C-16 (to Pkr signaling). Usp18, Ifi44, Oas1g, Isg15, and Ifnb1 RNA levels were measured by RT-qPCR. p values were determined by one-way or two-way ANOVA followed by Tukey’s HSD test (in c, d). e, f Interaction between CSpV1-dsRNAs and components in the Rig-I/Mavs/Pkr pathways in IEC4.1 cells following infection or CSpV1-dsRNA transfection. Cells were exposed to C. parvum or transfected with CSpV1-dsRNAs for 24 h. Cell lysates were collected for RNA-protein interaction measurement using RNA immunoprecipitation assay. For infected cells, a parasite RNA, Cgd7_Flc_0990, was used for control. p values were determined by two-tailed unpaired Student’s t-test (in e, f). Data (in c–f) are from three biological replicates and presented as mean values ± SD. g Model of CSpV1-dsRNAs to trigger type I IFN signaling in host cells. CSpV1-dsRNAs can be recognized by RIG-I and PKR in infected cells, resulting in activation of the Pkr and Rig-I/Mdv5/Sting signaling pathways and subsequently, activation of type I IFN signaling. Source data are provided as a Source Data file.