The Capsid Protein VP1 of Coxsackievirus B Induces Cell Cycle Arrest by Up-Regulating Heat Shock Protein 70

Figures

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  FIGURE 1. VP1 of CVB3 induces cell cycle arrest at G1 phase. (A,B) HeLa cells were transfected with pEGFP-VP1 or pEGFP-C1 for 24 h. The expression of VP1 was determined by fluorescence microscopy (A, lower panel; arrow heads indicate the nuclei). The relative fluorescence intensity of EGFP was quantified (B). The RNA level of VP1 was determined by RT-PCR (C). (D–F) HeLa cells were transfected with pEGFP-VP1 for 24 h. Control cells were transfected with pEGFP-C1. Cell cycle was analyzed by flow cytometry (D). Cell population distributed in each phase of the cell cycle was compared between the cells expressing VP1 and control cells (E,F). n = 3. *P < 0.05, ∗∗∗P < 0.01.

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  FIGURE 2. VP1 of CVB3 induces G1 arrest in cadrdiomyocytes. (A) Cardiomyocytes were isolated from the hearts of neonatal Balb/c mice. Cells were transfected with pEGFP-C1 or pEGFP-VP1 for 24 h and viewed by fluorescence microscope. (B,C) Cell cycle was determined by flow cytometry. (D,E) Cell population distributed in each phase of the cell cycle was compared between the cells expressing VP1 and control cells. n = 3. *P < 0.05.

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  FIGURE 3. VP1 dysregulates the expression of cyclin E and p27kip1. (A,B) HeLa cells were transfected with pEGFP-C1 or pEGFP-VP1 for 24 h. Cyclin E and p27kip1 were determined by Western blotting. n = 3. *P < 0.05, ∗∗P < 0.01.

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  FIGURE 4. Up-regulated Hsp70 in the cells expressing VP1 results in the dysregulation of cyclin E and p27kip1. HeLa cells were transfected with pEGFP-C1 or pEGFP-VP1 for 24 h. (A) The expression of Hsp70 was determined by RNA-sequencing. (B,C) The expression of Hsp70 was verified by Western blotting. (D,E) HeLa cells were transfected with si-Hsp70 for 24 h. Cell lysate was prepared and cyclin E, p27kip1, and Hsp70 were determined by Western blotting. (F) HeLa cells were transfected with pEGFP-C1 or pEGFP-VP1 for 24 h, HSF1 and p(S326)-HSF1 were determined by Western blotting. n = 3. *P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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  FIGURE 5. Coxsackievirus B3 infection induces G1 arrest. HeLa cells were cultured in 6-well plate to 60% confluence and infected with CVB3 at 1 MOI for 24 h. Mock-infected cells were set as control. (A) Cell cycle was determined by flow cytometry at 24 h of post-infection. (B) Cell population distributed in each phase of the cell cycle was calculated. (C) Cell population was compared between mock-infected and CVB3-infected cells. (D) The 3Dpol of CVB3 was determined by Western blotting. n = 3. ∗∗P < 0.01.

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  FIGURE 6. Coxsackievirus B3 infection dysregulates the expression of Hsp70, cyclin E, and p27kip1. (A,B) HeLa cells were cultured in 6-well plate to 60% confluence and infected or mock-infected with CVB3 at 1 MOI for 24 h. Cell lysates were prepared and subjected to the analysis of Western blotting. (C) HeLa cells were transfected with si-Hsp70 for 24 h and infected with CVB3 at MOI of 1 for 12 h. Cell lysates were prepared and subjected to the analysis of Western blotting. n = 3. *P < 0.05. si-Control: Control si-RNA.

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  FIGURE 7. G1 arrest facilitates the replication of CVB3. (A) HeLa cells were synchronized at G1 by theaddition of thymidine and cells were infected with CVB3. (B) Cell cycle was analyzed by flow cytometry. (C) Cell population distributed in the various phases of cell cycle was calculated. (D) Cell population in G1 phase was compared between CVB3-infected cells with and without treatment of thymidine. (E) Viral RNA of CVB3 was determined by RT-qPCR. n = 3. *P < 0.05, ∗∗∗P < 0.001. n.s.: no significant.