Figure 1.
3Dpol associates with the nuclear protein Prp8.
(A) Identification of potential 3Dpol-interacting host proteins. The cell lysates for IP were harvested from EV71 40 MOI-infected RD cells at 6 h.p.i. and treated with the 3Dpol monoclonal antibody or untreated as a negative control. The proteins that interacted with 3Dpol were pulled down using an anti-3Dpol antibody, along with protein A-Sepharose, and detected by 1D SDS-PAGE and silver staining. (B) 3Dpol interacts with 5 components of U5 snRNPs, including Prp8, Brr2, Snu114, Prp6, and SNRNP40. The interaction of EV71 3Dpol and the nuclear protein U5 snRNPs was further confirmed by Co-IP and WB assays. The lysates harvested from mock- or EV71 40 MOI-infected RD cells at 2 to 8 h.p.i. were treated with RNase A (10 µg/ml) and immunoprecipitated using an anti-3Dpol antibody. The 5 components of the U5 snRNPs that interacted with 3Dpol were detected using a WB assay. The input samples were verified in the presence of 3Dpol and the five components of the U5 snRNPs in the lysates. Actin served as an internal control. (C) The core spliceosome splicing factor Prp8 can also pull down 3Dpol and 3CD. EV71-infected RD cell lysates from 2 to 8 h.p.i. were treated with RNase A (10 µg/ml) and incubated with antibodies against the Prp8 probe. After the IP assay, 3Dpol and 3CD were analyzed using WB with an anti-3Dpol antibody. (D) 3Dpol associates with the C-terminal domain of Prp8 containing the Jab1/MPN region. The functional domain architecture of human Prp8 is shown (upper panel). HEK293T cells were transfected with plasmids encoding full-length FLAG-3Dpol (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24), various truncated forms of HA-Prp8 (lanes 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, and 24), and empty vectors (lanes 1, 5, 9, 13, 17, and 21). At 48 h after transfection, the lysates were treated with RNase A (10 µg/ml) and immunoprecipitated with antibodies against FLAG. The truncated form of Prp8 that interacted with 3Dpol was detected by WB using an antibody against HA. (E) The C-terminal domain of Prp8 interacts with the fingers domain of 3Dpol. The functional domain architecture of EV71 3Dpol is shown (upper panel). HEK293T cells were transfected with plasmids encoding HA-Prp8-2094-2335 (lanes 3, 4, 7, 8, 11, 12, 15, and 16), various truncated forms of FLAG-3Dpol (lanes 2, 4, 6, 8, 10, 12, 14, and 16), and empty vectors (lanes 1, 5, 9, and 13). The various truncated forms of 3Dpol were pulled down by IP with an anti-FLAG antibody. The C-terminal domain containing the Jab1/MPN region of Prp8, which interacts with the truncated form of FLAG-3Dpol, was detected with an anti-HA antibody in a WB assay.
Table 1.
Potential protein targets of EV71 3Dpol were identified by MALDI-TOF MS analysis.
Figure 2.
3Dpol and Prp8 are colocalized in the nucleus at 4 h.p.i.
(A) The 3Dpol-Prp8 association is localized in the nucleus at 4 h.p.i. Mock- or EV71 40 MOI-infected RD cells were fixed and stained using antibodies against EV71 3Dpol (green color) and Prp8 (red color) at 2, 4, 6, and 8 h.p.i. The nuclei of RD cells were stained with Hoechst 33258 dye (blue color), and the merged images show the 3Dpol and Prp8 immunofluorescence signals. All immunofluorescence images were detected by confocal microscopy. Scale bar, 10 and 20 µm. (B) 3CD, 3Dpol, and Prp8 appear in the nuclei of infected cells at 4 h.p.i. The cytoplasmic (C) and nuclear (N) fractions of EV71-infected RD cells at 2 to 4 h.p.i were extracted and loaded with the same percent-volume for SDS-PAGE. EV71 3CD, 3Dpol, and Prp8 were detected using anti-3Dpol and Prp8 antibodies in a WB assay. GAPDH and Lamin A/C were detected as cytoplasmic and nuclear protein controls, respectively. (C) EV71 3Dpol enters the nucleus through the KKKD amino acids of the NLS. FLAG-tagged constructs of 3Dpol containing the FLAG-tagged wt 126–129 aa NLS (KKKD) and mutant NLS (AAAA) were used to map the NLS on EV71 3Dpol. RD cells were transfected with these plasmids expressing FLAG-3Dpol-wt or FLAG-3Dpol-mut for 48 h and then stained using antibodies against FLAG (green color). The nuclei were stained with Hoechst 33258 dye (blue color). The immunofluorescence was visualized by confocal microscopy. Scale bar, 10 and 20 µm.
Figure 3.
The EV71 and PV 3Dpol interfere with the splicing process and inhibit mRNA synthesis.
(A) Recombinant EV71 3Dpol inhibits mature mRNA production. An in vitro splicing assay was performed for 90 min using 32P-labeled PIP85a pre-mRNA as the substrate, nuclear extracts of HeLa cells, and varying amounts of purified recombinant 3Dpol. The autoradiogram revealed the presence of different radioactive RNA forms, including pre-mRNA, the lariat form, excised intron, mature mRNA, and exon1. (B) Recombinant EV71 3Dpol stops the splicing process in the lariat form. The in vitro splicing substrate, 32P-labeled PIP85a pre-mRNA, was incubated with mock- or EV71 3Dpol recombinant protein-containing nuclear extracts for varying time periods. The autoradiogram shows the different forms of RNAs in the splicing reaction. (C) Recombinant EV71 and PV 3Dpol inhibit the synthesis of mature mRNA. The in vitro splicing assay was performed using the same conditions described above, including a protein concentration of 4 µM and a reaction time of 90 min, with recombinant 3Dpol proteins from EV71, PV, CVB3, and HRV16. (D) The EV71 and PV 3Dpol proteins directly associate with the C-terminal domain of Prp8. In vitro pull-down assay, a total of 5 µg of bacterially purified His+-3Dpol from EV71, PV, CVB3, or HRV16 was mixed with 5 µg of the GST-Prp8-C-terminal domain fusion protein for 90-min reaction time, followed by GST pull-down and WB assays.
Figure 4.
3Dpol 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 3Dpol in infected cells were detected using anti-HA and anti-EV71 3Dpol 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) 3Dpol inhibits cellular pre-mRNA splicing by interacting with Prp8. RD cells were transfected with constructs encoding FLAG-tagged 3Dpol (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 3Dpol 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.
Figure 5.
RIP-seq of the pre-mRNA trapped by the Prp8-3Dpol complexes.
(A) Procedural diagram for the RIP assays. The schematic shows the experimental procedure for characterizing RNA from Prp8 or Prp8-3Dpol complexes by IP. (B) The Prp8 antibody pulls down the Prp8 and Prp8-3Dpol complexes. In the WB analysis, the pulled down Prp8 and Prp8-3Dpol complexes demonstrated the efficiency of Prp8 IP and the interaction between Prp8 and 3Dpol. (C) A flow chart for the selection of targeted RNAs from the sequencing data. The schematic shows the experimental procedure for characterizing RNAs from Prp8 or Prp8-3Dpol complexes. (D) The inhibition of the pre-mRNA splicing in intracellular targeted cyclin D3 and PDGF. The increase in pre-mRNA and decrease in mRNA for intracellular cyclin D3 and PDGF in EV71-infected cells at 4–6 h.p.i. validated our RIP-Seq analysis. Error bars, mean ± SD (n = 3). The statistical significance was analyzed using a t test. ***p<0.001; **p<0.01; *p<0.05.
Table 2.
The differentially expressed transcripts were classed into groups according to functional annotations from the KEGG pathways.
Figure 6.
Schematic model of 3Dpol-mediated inhibition of the cellular splicing by targeting Prp8 in the nucleus.
3Dpol primarily performs viral RNA replication in the host cytoplasm, but partially 3Dpol also enters the nucleus and interacts with the core splicing factor Prp8, which interferes with the function of Prp8 in the C1-complex. The interference of Prp8 function inhibits the second step of the splicing process and results in the accumulation of the lariat form and a reduction in mRNA synthesis.