Fig 1.
CVB3 VP1 affects cell proliferation and blocks cell cycle at the G1/S phase.
(A) The numbers of adherent HPAC cells in different groups were counted using Image Pro Plus (IPP) software. (B) HPAC cells were transfected by pBud-VP1 and pBud plasmids, and the cell proliferation was evaluated using the CCK-8 assay. (C) The activity of DNA replication was examined using EdU incorporation in HPAC cells. (D) G1/S phase arrest induced by VP1 transfection. HPAC cells were treated with 36-hour plasmid transfection (pBud and pBud-VP1) within the treatment of double-thymidine, and collected at 0, 6 and 15 hpi separately, then cells were analyzed by flow cytometry. Values are shown as the mean ± SEM of three independent experiments. (*P<0.05, **P<0.01).
Fig 2.
CVB3 VP1 may directly interact with MAT1.
(A) Plasmids pGBKT7-VP1 and pGADT7-MAT1 were co-transformed with yeast strain AH109, and the positive result selected on high-stringency medium (SD/-Ade/-His/-Leu/-Trp/X-α-Gal). Interaction is reflected by blue color, while white colonies suggest no interaction. (B) the VP1-MAT1 interaction identified by transcription-translation system in vitro. Plasmids HA-MAT1 and c-Myc-VP1 were labeled with 35S-Methionine, and immunoprecipitated with antibody against c-Myc; the binding proteins with 35S-Methionine were analyzed by 10% SDS-PAGE and autoradiography. (C) Co-immunoprecipitation detected the interaction of VP1 and MAT1 in CVB3 infected cells. At 3 hours post infection, the cells lysates of mock and CVB3 infected cells were incubated with or without monoclonal antibody against MAT1 or irrelevant IgG affinity gel, and analyzed by western blotting with the specific antibodies after immunoprecipitation. Original blots are shown in S16 Fig for statistical analysis. (D) MAT1 and VP1 intracellular localization in CVB3 infected HPAC cells. Representative confocal immunofluorescence microscopic images of MAT1 and VP1 stained with rabbit anti-MAT1 (green) and mouse anti-VP1 antibodies (red), respectively, the MAT1 and VP1 images were also merged; the nuclei are labeled with DAPI. Scale bar = 10 μm. Values are shown as the mean ± SEM of three independent experiments. (*P<0.05, **P<0.01).
Fig 3.
CVB3 VP1 impairs the assembly of a functional CAK complex.
(A) Confocal microscopy analysis of the abundance of VP1 and MAT1 in HPAC cells, Cells were detected with monoclonal antibodies to MAT1 (Alexa-488; green) and polyclonal anti-VP1 (Alexa-647; red), and counterstained with DAPI to show the nucleus. The MAT1 and VP1 images were merged. Scale bar: 10 μm. The integrated optical density (IOD) represented the fluorescent signal was obtained by statistical analysis of 20 fields (5–8 cells in each field) randomly selected using Image J software. (B) Immunoblot analysis of the nuclear-localized accumulation of MAT1, CDK7, Cyclin H in CVB3 infected, VP1 and pBud transfected HPAC cells by specific antibodies. Original blots are shown in S17 Fig for statistical analysis. (C) CVB3 VP1 induces ubiquitination-proteolysis of MAT1. The cell lysates of pBud-VP1 and pBud transfected cells were incubated with monoclonal antibody against MAT1 with 0, 3, 6, 9 hrs. The generated blot was then analyzed by immunoblotting with anti-ubiquitin antibody. Original blots are shown in S18 Fig for statistical analysis. (D) CVB3 VP1 induces ubiquitination-proteolysis of CDK7. Original blots are shown in S19 Fig for statistical analysis. Values are shown as the mean ± SEM of three independent experiments. (*P<0.05, **P<0.01).
Fig 4.
CVB3 VP1 alters the subcellular colocalization of MAT1 and CDK7 in G1/S phase.
(A) Probe showing the time points when the cells were mainly at G1 and S phases by flow cytometry. The cells were synchronized using double thymidine block treatment, and released every hour. (B) Patterns of infection and transfection time points. The cells are synchronized at G1 (46 hrs) and S phase (51 hrs) by thymidine double blockade. (C and D) The alteration in spatial localization of MAT1 and CDK7 by thymidine double blockade at G1 and S phases, respectively. The cells were transfected with pBud-VP1 and infected with CVB3, and then imaged using confocal microscopy. Confocal image shows MAT1 (green), CDK7 (red), DAPI (blue) and merged views. Scale bar: 10 μm. (E and F) After nuclear and cytoplasmic extraction, the expression of MAT1 and CDK7 in G1 and S phase of cells infected with CVB3 or transfected with pBud-VP1 plasmid were detected. Original blots are shown in S20 and S21 Figs for statistical analysis. Values are shown as the mean ± SEM of three independent experiments. (*P<0.05, **P<0.01).
Fig 5.
CVB3 VP1 could suppress CAK activity in vivo.
(A) Time-course analysis of the accumulation of phosphorylated/nonphosphorylated CDK2 and CDK4 by indicated antibodies. Original blots are shown in S22 Fig for statistical analysis. (B) The expression level and phosphorylation of RNA Pol II CTD were detected by antibodies against RNA Pol II CTD and phospho-RNA Pol II CTD. CVB3 infection was the positive control. Original blots are shown in S23 Fig for statistical analysis. (C) Immunoblot analysis of the accumulation of phosphorylated/nonphosphorylated pRb with phospho-pRb Ser 780, 795, 807/811 antibodies. CVB3 infection was the positive control. Original blots are shown in S24 Fig for statistical analysis. (D) The abundance of RNA Pol II CTD, phospho-RNA Pol II CTD and CDK7 were analyzed by Western blot, and this assay was performed at 48 hours after transfecting various plasmids. Original blots are shown in S25 Fig for statistical analysis. (E) Western blotting analysis of the expression of phosphorylated/non-phosphorylated pRb and CDK7 with phospho-pRb Ser 780, 795, 807/811 and CDK7 antibodies. Original blots are shown in S26 Fig for statistical analysis.
Fig 6.
CVB3 VP1 inhibited the activity of CDK4/6 and Rb phosphorylation of the CDK-Rb signaling pathway in the G1/S transition.
Abemaciclib (5 mg/ml stock solution in DMSO) was diluted to a final concentration of 10 μM in culture medium to treat cells. (A) Abemaciclib (Abema) further inhibits cell proliferation with VP1. An EdU assay was used to analyze the cell proliferation of Mock and pBud-VP1 transfected HPAC cells with or without abemaciclib treatment. (B) Abemaciclib further inhibits cell viability with VP1. A CCK-8 assay was used to analyze the cell viability of Mock and pBud-VP1 transfected HPAC cells with or without abemaciclib treatment. (C) Abemaciclib further inhibits phosphorylated pRb with VP1. Western blotting analysis of the expression of phosphorylated/nonphosphorylated pRb with phosphor-pRb Ser 780, 807/811 antibodies. Original blots are shown in S27 Fig for statistical analysis. (D) CDK4/6 inhibitor further inhibits the cell cycle based on pBud-VP1. HPAC cells were treated with pBud and pBud-VP1 transfection within the treatment of the double-thymidine and abemaciclib, then released and separately collected at 12 and 15 hpi; the right figure is flow cytometric results. Values are shown as the mean ± SEM of three independent experiments. (*P<0.05, **P<0.01).
Fig 7.
Mapping the minimum domain of VP1 interaction with MAT1.
(A) Construction of a series of VP1 truncation/deletion mutants. The dark blue box is the full-length sequence encoded by VP1, the gray boxes are the VP1 deletion mutants that cannot interact with MAT1, the light blue boxes are the VP1 mutants that were detected to interact with MAT1, the D8 (red box) was the minimum domain of VP1 for the interaction (768–859 aa) in the VP1 C-terminus. (B) The interaction between VP1-D8 and MAT1 arrested cell cycle at the G1/S phase. pBud, pBud-VP1, pBud-VP1-D4 and pBud-VP1-D8 transfected cells for 48 h, and the cells were harvested and analyzed by flow cytometry. (C) VP1-D8 transfection down-regulated the expression of MAT1. Immunoblot analysis of the abundance of MAT1 in pBud, VP1, VP1-D4 and VP1-D8 transfected cells. Original blots are shown in S28 Fig for statistical analysis. (D) VP1-D8 transfection down-regulated the phosphorylation of CDK2 and CDK4. Western blot analysis of the accumulation of phosphorylated/nonphosphorylated CDK2 and CDK4 in different groups. Original blots are shown in S29 Fig for statistical analysis. (E) The accumulation of Pol II CTD, phospho-RNA Pol II CTD and β-actin were analyzed by Western blot. Original blots are shown in S30 Fig for statistical analysis. (F) Western blotting analysis of the expression of phosphorylated/nonphosphorylated pRb with specific antibodies. Original blots are shown in S31 Fig for statistical analysis. Values are shown as the mean ± SEM of three independent experiments. (*P<0.05, **P<0.01).
Fig 8.
A schematic diagram representing how CVB3 VP1 inhibits cell proliferation via impairing the CAK complex through its interaction with MAT1.
As CVB3 invades HPAC and Hela cells, CVB3 VP1 interacts competitively with MAT1, leading to the degradation of CDK7, thus the assembly of the CAK complex is inhibited, and the activity of CAK is simultaneously inhibited. CAK affects the activity of cyclin-dependent kinases CDK2 and CDK4, and then inhibits the phosphorylation level of pocket Rb protein, inducing the persistent binding of Rb and E2F protein, and finally inhibits the expression of downstream transcription factors by E2F and cell cycle arrest occurs in the G1 phase. CAK also affects the phosphorylation of its substrate RNA Pol II CTD Ser 5, which eventually leads to the inability of RNA Pol II to participate in cell transcription and cell cycle arrest at the G1 phase.