Human Enterovirus 68 Interferes with the Host Cell Cycle to Facilitate Viral Production

Figure 1. Different cell cycle stages have profound effects on EV-D68 replication. The effects of cell cycle synchronization on EV-D68 are shown for G0/G1 arrest (A–D), S phase arrest (E–H), and G2/M arrest (I–L). (A,E,I) Flow diagram of how RD cells were treated with serum starvation (starved) for G0/G1 synchronization (A), with thymidine (thymi) for S synchronization (E), or with nocodazole (noco) for G2/M synchronization (I). The top diagram in each panel shows the strategy for the control group, and the bottom panel shows the strategy for cell cycle synchronization. (B,F,J) Cell-cycle profiles were determined by flow cytometry after G0/G1, S, and G2/M synchronization with serum starvation, thymidine, and nocodazole treatment, respectively. Histograms below show the percentage of cells in each phase of the cell cycle as analyzed by the ModFit LT program. (C,G,K) Levels of intracellular EV-D68 Fermon strain RNA were detected in RD cells after cell cycle synchronization by quantitative real-time PCR. The results were standardized to GAPDH mRNA expression and normalized to 1.0 in mock-infected cells. (D,H,I) Progeny viruses in the supernatants were titrated using RD cells. A relative…

Figure 2. EV-D68 infection induces G0/G1 arrest. (A,C,D) RD cells were infected with EV-D68 Fermon (A), US/MO/14-18947 (C) or US/KY/14-18953 (D) at an MOI of 0.8 or mock for 24 h for analysis of cell cycle status. Left: Cell-cycle profiles were determined by flow cytometry. Middle: The histograms show the percentage of cells in each phase of the cell cycle in mock-infected and EV-D68-infected cells. Right: The ratio of G0/G1 to G2/M is shown. (B) The virulence of Fermon, US/KY/14-18953 and US/MO/14-18947strains in infected RD cells was assessed 24 h post-infection with virus at MOI 0.8. Values are expressed as the TCID50/mL of the progeny viruses in the supernatant. The results represent the mean ± S.D of three independent experiments. **P < 0.01 and ***P < 0.001.

Figure 3. EV-D68 infection prevents cell exit from G0/G1 into S phase and promotes G2/M to G0/G1 transition. (A) RD cells were serum-starved for 24 h and then mock-infected (mock) or infected with EV-D68 Fermon strain (infected) at an MOI of 0.8. After 2 h of virus adsorption, the cells were treated with medium without FBS for 18 h, followed by medium containing 10% FBS. Top panel: The cell cycle profiles were determined by flow cytometry at 0, 18, 24, and 30 h post-infection. Bottom panel: The histograms show the percentage of cells in each phase of the cell cycle. (B) G0/G1 and S phase-related cell cycle proteins were analyzed by western blot analysis. RD cells were mock-infected (m) or infected with EV-D68 Fermon strain at an MOI of 0.8 (i) for 0, 16, 20, 24, and 28 h. Histone is shown as a loading control. Results are representative of three independent experiments. (C) At 24 h post-infection, mRNA levels of CDK2, cyclinE1, CDK4, CDK6, and cyclinD were evaluated in mock infected (mock) and EV-D68 infected (infected) cells by quantitative real-time PCR. The results are standardized to GAPDH and normalized to 1.0 in mock-infected cells. (D) RD cells were treated with 25 ng/mL nocodazole…

Figure 4. The non-structural protein 3D and 3C of EV-D68 mediates cell cycle alterations. (A,C) Effect of 3D and 3C on cell cycle progression. Left panel: The distribution of cell cycle in 293T cells was analyzed at 36 h after transfection with 2 μg of VR1012-3D-HA (3D), VR1012-3C-HA (3C) or the corresponding control vector VR1012 (mock). Right panel: The ratio of cells in G0/G1 to G2/M at 36 h after transfection with 2 μg of VR1012-3D-HA (3D), VR1012-3C-HA (3C) or the corresponding control vector VR1012 (mock). (B,D) The cell cycle distribution in 293T cells was analyzed at 36 h after transfection with 0, 0.5, 1, or 2 μg plasmid as indicated. Left panel: The histograms show the percentage of each phase in the cell cycle. Right panel: The ratios of cells in G0/G1 to G2/M. (E,F) The expression of cell cycle-related proteins after transfection of 293T cells with 3D, 3C or corresponding vector (mock) was assessed by Western blot analysis at 36 h. Histone is shown as a loading control. (G) Model for the distinct effects of the non-structural proteins 3D and 3C in cell cycle arrest caused by EV-D68. The results indicate the mean ± S.D of three independent experiments. **P < 0.01 and ***P < 0.001.

Figure 5. G0/G1-phase synchronization has different effect on the replication of EV-A71 and EV-D68. RD cells infected with EV-D68 Fermon strain (A,C,E) or EV-A71 (B,D,F) at an MOI of 0.8 and the effects of synchronization by starvation were assessed. (A,B) RD cells were serum-starved for 48 h to synchronize cells in G0/G1 phase. The cell-cycle distribution was then detected by flow cytometry. The histograms showed the percentage of each phase in the cell cycle. (C,D) At 18 h post-infection, intracellular EV-D68 and EV-A71 RNA levels were assessed in control medium (con+infected)-treated or no serum (starved+infected)-treated RD cells by quantitative real-time PCR. The results were standardized using GAPDH mRNA as a control and normalized to 1.0 in mock-infected cells. (E,F) At 24 h post-infection, The TCID50/mL was shown through titrating the progeny viruses of EV-D68 or EV-A71 in the supernatants with RD cells. The results represent the mean ± S.D of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6. Synchronization in the G2/M phase inhibits the replication of EV-D68 and SV-A71. (A–C) RD cells were treated with or without 25 ng/mL nocodazole (noco) for 24 h, infected with EV-D68 Fermon (FER), US/MO/14-18947 (MO) or US/KY/14-18953 (KY) strains at an MOI of 0.8 for 2 h, and then treated again with or without 25 ng/mL nocodazole for synchronization. (A) At 24 h post-infection, intracellular EV-D68 RNA levels were detected by quantitative real-time PCR. The results were standardized using GAPDH mRNA as a control and normalized to 1.0 in mock-infected cells. (B) At 24 h post-infection, the TCID50/ml of the progeny viruses was determined. (C–G) RD cells were infected with EV-D68 strains US/KY/14-18953 or EV-A71 at an MOI of 0.8 for 2 h, and then treated with 2 μM Pseudolaric acid B (PAB) for 24 h for G2/M synchronization. (C) RD cells were treated with 2 μM Pseudolaric acid B (PAB) for 24 h for G2/M synchronization. Top panel: Cell-cycle profiles were determined by flow cytometry. Bottom panel: The histograms show the percentage of cells in each phase of the cell cycle. (D,E) Intracellular EV-D68 or EV-A71 RNA levels were assessed in control medium (con+infected) or PAB containing…