Cytoplasmic Viral RNA-Dependent RNA Polymerase Disrupts the Intracellular Splicing Machinery by Entering the Nucleus and Interfering with Prp8

<div><p>The primary role of cytoplasmic viral RNA-dependent RNA polymerase (RdRp) is viral genome replication in the cellular cytoplasm. However, picornaviral RdRp denoted 3D polymerase (3D^pol) also enters the host nucleus, where its function remains unclear. In this study, we describe a novel mechanism of viral attack in which 3D^pol enters the nucleus through the nuclear localization signal (NLS) and targets the pre-mRNA processing factor 8 (Prp8) to block pre-mRNA splicing and mRNA synthesis. The fingers domain of 3D^pol associates with the C-terminal region of Prp8, which contains the Jab1/MPN domain, and interferes in the second catalytic step, resulting in the accumulation of the lariat form of the splicing intermediate. Endogenous pre-mRNAs trapped by the Prp8-3D^pol complex in enterovirus-infected cells were identified and classed into groups associated with cell growth, proliferation, and differentiation. Our results suggest that picornaviral RdRp disrupts pre-mRNA splicing processes, that differs from viral protease shutting off cellular transcription and translation which contributes to the pathogenesis of viral infection.</p></div

RIP-seq of the pre-mRNA trapped by the Prp8-3D^pol complexes.

<p>(A) Procedural diagram for the RIP assays. The schematic shows the experimental procedure for characterizing RNA from Prp8 or Prp8-3D^pol complexes by IP. (B) The Prp8 antibody pulls down the Prp8 and Prp8-3D^pol complexes. In the WB analysis, the pulled down Prp8 and Prp8-3D^pol complexes demonstrated the efficiency of Prp8 IP and the interaction between Prp8 and 3D^pol. (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 Prp8-3D^pol 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 <i>t</i> test. ^***p<0.001; ^**p<0.01; ^*p<0.05.</p

Schematic model of 3D^pol-mediated inhibition of the cellular splicing by targeting Prp8 in the nucleus.

<p>3D^pol primarily performs viral RNA replication in the host cytoplasm, but partially 3D^pol 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.</p

The EV71 and PV 3D^pol interfere with the splicing process and inhibit mRNA synthesis.

<p>(A) Recombinant EV71 3D^pol inhibits mature mRNA production. An <i>in vitro</i> 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 3D^pol. 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 3D^pol stops the splicing process in the lariat form. The <i>in vitro</i> splicing substrate, ³²P-labeled PIP85a pre-mRNA, was incubated with mock- or EV71 3D^pol 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 3D^pol inhibit the synthesis of mature mRNA. The <i>in vitro</i> 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 3D^pol proteins from EV71, PV, CVB3, and HRV16. (D) The EV71 and PV 3D^pol proteins directly associate with the C-terminal domain of Prp8. <i>In vitro</i> pull-down assay, a total of 5 µg of bacterially purified His<b>⁺</b>-3D^pol 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.</p

3D^pol and Prp8 are colocalized in the nucleus at 4 h.p.i.

<p>(A) The 3D^pol-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 3D^pol (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 3D^pol and Prp8 immunofluorescence signals. All immunofluorescence images were detected by confocal microscopy. Scale bar, 10 and 20 µm. (B) 3CD, 3D^pol, 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, 3D^pol, and Prp8 were detected using anti-3D^pol and Prp8 antibodies in a WB assay. GAPDH and Lamin A/C were detected as cytoplasmic and nuclear protein controls, respectively. (C) EV71 3D^pol enters the nucleus through the KKKD amino acids of the NLS. FLAG-tagged constructs of 3D^pol containing the FLAG-tagged wt 126–129 aa NLS (KKKD) and mutant NLS (AAAA) were used to map the NLS on EV71 3D^pol. RD cells were transfected with these plasmids expressing FLAG-3D^pol-wt or FLAG-3D^pol-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.</p

The differentially expressed transcripts were classed into groups according to functional annotations from the KEGG pathways.

<p>Footnotes.</p><p>Term: gene set name.</p><p>Count: number of genes associated with this gene set.</p><p>Percentage (%): gene associated with this gene set/total number of query genes.</p><p>P-value: modified Fisher Exact P-value.</p><p>List Total: number of genes in your query list mapped to any gene set in this ontology.</p><p>Pop Hits: number of genes annotated to this gene set on the background list.</p><p>Pop Total: number of genes on the background list mapped to any gene set in this ontology.</p

Potential protein targets of EV71 3D^pol were identified by MALDI-TOF MS analysis.

<p>Footnotes:</p><p>The score of the above-mentioned proteins was greater than 50. The band numbered 1, 4–5, 7–8, 11–13, 18–22, 25, 28–31, 34–37 did not achieve a significant score for protein identification and were not listed in the Table.</p><p>The sequence coverage for these proteins determined by MALDI-TOF analysis is also indicated.</p

3D^pol associates with the nuclear protein Prp8.

<p>(A) Identification of potential 3D^pol-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 3D^pol monoclonal antibody or untreated as a negative control. The proteins that interacted with 3D^pol were pulled down using an anti-3D^pol antibody, along with protein A-Sepharose, and detected by 1D SDS-PAGE and silver staining. (B) 3D^pol interacts with 5 components of U5 snRNPs, including Prp8, Brr2, Snu114, Prp6, and SNRNP40. The interaction of EV71 3D^pol 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-3D^pol antibody. The 5 components of the U5 snRNPs that interacted with 3D^pol were detected using a WB assay. The input samples were verified in the presence of 3D^pol 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 3D^pol 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, 3D^pol and 3CD were analyzed using WB with an anti-3D^pol antibody. (D) 3D^pol 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-3D^pol (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 3D^pol was detected by WB using an antibody against HA. (E) The C-terminal domain of Prp8 interacts with the fingers domain of 3D^pol. The functional domain architecture of EV71 3D^pol 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-3D^pol (lanes 2, 4, 6, 8, 10, 12, 14, and 16), and empty vectors (lanes 1, 5, 9, and 13). The various truncated forms of 3D^pol 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-3D^pol, was detected with an anti-HA antibody in a WB assay.</p

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

<p>(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 <i>t</i> test. ^***p<0.001; ^**p<0.01.</p