Figure 4. 3D^pol affects cellular pre-mRNA splicing by interacting with Prp8.

(A) EV71 inhibits cellular pre-mRNA splicing by interacting with Prp8. RD cells were transfected with pCMV-HA (lanes 1, 2, 5, and 6) or the vector encoding HA-tagged Prp8 (lanes 3, 4, 7, and 8). After 24 h, the exogenous reporter pSV40-CAT(In1), which encodes chloramphenicol acetyl transferase inserted by human β-globin intron 1, was transfected into all of the samples for 24 h. The total RNA obtained from RD cells was subsequently harvested after EV71 40 MOI infection at 2 h.p.i. (lanes 2 and 4) and 4 h.p.i. (lanes 6 and 8) for RT-qPCR. The fold changes in the amount of pre-mRNA and mRNA were calculated. The overexpression of HA-tagged Prp8 and the level of viral 3D^pol in infected cells were detected using anti-HA and anti-EV71 3D^pol antibodies, respectively, in a WB assay. (B) To confirm the effects of EV71 on endogenous splicing, RNAs were isolated from EV71-infected cells at 2 to 4 h.p.i. and evaluated with a specific primer for nucleolin by RT-qPCR. (C) 3D^pol inhibits cellular pre-mRNA splicing by interacting with Prp8. RD cells were transfected with constructs encoding FLAG-tagged 3D^pol (lanes 3 and 4) or HA-tagged Prp8 (lanes 2 and 4). The vectors pFLAG-CMV2 and pCMV-HA were used as negative controls (lane 1). The exogenous reporter pSV40-CAT(In1) was transfected into all of the samples for 24 h, and the total RNA obtained was subsequently harvested from RD cells for RT-qPCR. The fold changes in the amount of pre-mRNA and mRNA were calculated. In a WB assay, the overexpression of HA-tagged Prp8 and the level of FLAG-tagged 3D^pol were detected using anti-HA and anti-FLAG antibodies, respectively. Error bars, mean ± SD (n = 3). The statistical significance was analyzed using a t test. ^***p<0.001; ^**p<0.01.