Fig 1.
RV-infected MA104 cells get arrested in S/G2 phase in a strain-independent manner.
(A) Schematic representation of the procedure used for cell synchronization by double blocking with thymidine (Thy) and further RV-infection. (B) Flow cytometer histograms of synchronized MA104 cells NI (upper row) or infected with simian RV strain SA11 [MOI, 25 VFU/cell] (lower row) at 0, 2, 4, 6, 8, 10 and 12 hours post-release (hpr) from thymidine block. The histograms overlay the DNA content obtained by the Watson model being purple, yellow and green areas under the curve of G1, S, and G2 phase percentage values, respectively. The plots show the relative (S+G2)/G1 ratio comparing NI and RV-infected [MOI, 25 VFU/cell] cells at each indicated time post-release from thymidine. As indicated, cells were infected with simian SA11 strain (C), porcine OSU strain (D) and simian RRV strain (E). The relative (S+G2)/G1 ratio was calculated considering NI at 0 hpr as a value of 1. Data represent the mean ± SEM from three independent experiments (t-test, *p<0.05; ** p<0.01).
Fig 2.
RV-infected cells get arrested in S/G2 phase in a cell type-independent manner.
Flow cytometer histograms from double-thymidine synchronized MDCK (Madin-Darby canine kidney cells), U2OS (human bone osteosarcoma epithelial cells) and CV-1 (monkey kidney cells) cells infected with simian RV strain SA11 [MOI, 25 VFU/cell] and analyzed at 8, 12 and 14 hpr from thymidine. The selected time for analysis was dependent on a previous characterization of a whole replication cycle of each cell type. The histograms overlay the DNA content obtained by DJF mathematical model where purple, yellow and green areas under the curve correspond to the values of G1, S and G2 phases, respectively. An immunofluorescence picture, above each of the DNA content histograms, represents an immunostaining for viroplasms detection (mouse mAb anti-NSP5 [56], green) and stained for nuclei (DAPI, blue). Scale bar is 100μm. The plot, at the right of each panel, compares the percentage of the interphase stages (G1, S, and G2) from NI and SA11-infected cells at the indicated time post-release from thymidine. Each data correspond to the mean± SEM from three independent experiments. The SA11-infected cells compared with NI cells at the same time post-release; where ns, not significant p>0.05, (**) p<0.01 and (***) p<0.001 using two-tailed Student’s t-test.
Fig 3.
The cell cycle arrest induced by RV in S/G2 phase improves viral replication.
Unsynchronized and synchronized MA104 cells were infected with porcine RV strain OSU [25 VFU/cell] and analyzed at 0, 6 and 8 hpr from double thymidine block. (A) Representative flow cytometer histograms of unsynchronized (upper panel) and synchronized (lower panel) MA104 cells NI or infected with porcine RV OSU strain [MOI, 25 VFU/cell]. The histograms overlay the DNA content obtained by the DJF model where purple, yellow and green areas under the curve correspond to the values of G1, S and G2 phases, respectively. (B) The plot for relative (S+G2)/G1 ratio from comparing NI and OSU-infected unsynchronized and synchronized cells at 0 and 8 hpr from thymidine. The relative (S+G2)/G1 ratio was calculated considering synchronized NI at 0 hpr as a value of 1. Data represent the mean ± SEM from four independent experiments (**) p<0.01 and ns p>0.05). (C) Immunofluorescence of unsynchronized and synchronized cells NI and SA11 infected at 8 hpr from thymidine. The merged images are immunostaining for viroplasms detection (mouse mAb anti-NSP5, green) and nuclei staining (DAPI, blue). Scale bar is 100 μm. The number (D) and size (E) of viroplasms per cell plotted at 8 hpr from thymidine for unsynchronized and synchronized cells. The data represented the mean ± SEM of four independent experiments, t-test, (**) p<0.01; number >150 cells. (F) Distribution of the viroplasm frequency in unsynchronized and synchronized cells at 8 hpr. (G) Immunoblotting from a normalized number of unsynchronized (U, lanes 1 and 3) and synchronized (S, lanes 2 and 4) cells at 6 (lanes 1 and 2) and 8 hpr (lanes 3 and 4). VP2, VP4, VP6, NSP3 and NSP5 viral proteins were detected using anti-VP2, anti-VP5, anti-RV, anti-NSP3 and anti-NSP5 antibodies, respectively. Alpha-tubulin was used as loading control. (H) At 8 hpr, RV-infected unsynchronized and synchronized cells were count normalized, lysed and viral titer was determined. Data are expressed as the percentages of infectivity obtained from unsynchronized cells, which is considered as 100% of infectivity. The data is the mean ± SEM of three independent experiments, t-test, (*) p<0.05.
Fig 4.
RV depletes cyclin B1 and replication as a condition for the cell cycle arrest.
(A) Immunofluorescence of NI or OSU-infected [MOI, 25 VFU/cell] synchronized NSP5-EGFP/MA104 cells at 6 hpr. Cells were fixed in paraformaldehyde and immunostained for cyclin B1 (mouse mAb anti-cyclin B1, red) or PCNA (mouse mAb anti-PCNA, red), viroplasms detected with NSP5-EGFP (green) and nuclei stained with DAPI (blue). A merged image is presented in the right column. Scale bar is 10μm. (B) Immunoblotting of cell lysates from synchronized MA104 cells NI or OSU-infected [MOI, 25 VFU/cell] at 0 and 8 hpr from thymidine for the detection of cyclin A, cyclin B1, and NSP5. GAPDH is the loading control. The molecular weight markers are indicated. (C) Porcine OSU strain TLPs [MOI, 25 VFU/cell] inactivated with UV/psoralen (UV/AMT). Thymidine-synchronized MA104 cells were NI or infected with transcriptionally active (-UV/AMT) or inactive (+UV/AMT) virus. Cell cycle histograms for 0 hpr (top panel) and 6 hpr (bottom panel) are presented. The histograms overlay the DNA content obtained by DJF model, where purple, yellow and green areas under the curve correspond to the values of G1, S, and G2 phases, respectively. (D) The plot representing the relative (S+G2)/G1 ratio comparing NI cells with active (-UV/AMT) or inactive (+UV/AMT) TLPs at 6 hpr.
Fig 5.
Actin, MTs, and kinesin are involved in the RV arrest of the cell cycle.
(A) An experimental model targeting host pathways by specific drug inhibitors directly related to RV-arrest of the MA104 cell cycle. At 8 hpr, flow cytometer histograms from RV-infected synchronized cells compared with those from both NI and RV-infected cells after adding the drug at 30 minutes post-release from thymidine to determine a reverting drug for the RV-induced arrest at S/G2 phase. Thus, three conditions can be expected: (i) the drug intrinsically arrests in S-phase, (ii) the drug cannot interfere with RV-arrest and (iii) the drug reverts the RV-arrest in S-phase. (B) Plot showing the Log10 ratio of the drugs for either not affecting (white bars) or reverting (gray bars) RV cell cycle arrest. The drugs grouped into the major inhibitory physiological pathways such as cytoskeleton, the molecular motor, protein synthesis, proteasome, deacetylases and cyclin-dependent kinases. Each set of data corresponds to the average of two independent experiments. The concentration for each of the drugs used in this assay was: 10μM nocodazole, 10 μM cytochalasin B, 10μM monastrol, 50μM ciliobrevin D, 100μg/ml cycloheximide, 10μM MG132, 10μM lactacystin, 10μM UBEI-41, 10μM tubacin, 5mM Na-butyrate and 10μM purvalanol A.
Fig 6.
RV subverts kinesin motor Eg5.
(A) Immunofluorescence of NI or SA11-infected [MOI, 25 VFU/cell] synchronized MA104 cells at 8 hpr, for the visualization of Eg5 (anti-Eg5, red), viroplasms (anti-NSP5, green) and nucleus (DAPI, blue). Scale bar is 10 μm. (B) Eg5 perinuclear area determined for NI and SA11-infected synchronized MA104 cells at 8 hpr. Data represents the mean ± SEM (t-test, *** p<0.001, n≥30). (C) Immunoblotting of cell lysates at 0 and 8 hpr from NI and OSU-infected [MOI, 25 VFU/cell] synchronized MA104 cells. Viral infection detected with an anti-NSP5 specific antibody; phosphorylated Eg5 and Eg5 detected with rabbit anti-Eg5 (phosphoThr927) and mouse mAb anti-Eg5, respectively. GAPDH staining was used as loading control. Protein molecular weights are indicated. The phosphorylated-Eg5 positive control corresponds to cells treated post-synchronization with nocodazole (200ng/μl) [60].
Fig 7.
NSP3, NSP5, and VP2 play a role in the RV cell cycle arrest.
(A) Fluorescence microscopy of non-synchronized MA104-Fucci cells showing human Cdt1 (aa 30–120) fused to orange fluorescent monomeric Kusabira-Orange 2 (G1-mKO2) (red, top image), human Geminin (aa 1–110) fused to green fluorescent monomeric Azami-Green1 (S/G2/M-mAG, green)(middle image) and the fluorescence merge with bright field (bottom image). Red, yellow and green arrowheads indicate cells in early/late G1, G1/S, and S/G2/M phases respectively. Scale bar is 100μm. (B) Density plot of NI or OSU-infected [MOI, 25 VFU/cell] synchronized MA104-Fucci cell populations at 0 and 8 hpr. The population percentages, discriminated by its mAG and mKO2 fluorescence intensities, were gated for late G1, early G1, G1/S and S/G2/M. (C) Interphase stages plot of NI and OSU-infected synchronized MA104-Fucci cells at 0 and 8 hpr. Data are represented as the mean ± SEM (t-test, ***p<0.001, n = 3). (D) Experimental model for determination of RV protein(s) responsible for the cell cycle arrest. MA104-Fucci cells were transfected with RV proteins fused to CFP at their N- or C-terminus. Cells were harvested at 24 hpt and immediately analyzed by flow cytometry. The +CFP cells were gated, and interphase stages were analyzed using the Fucci system. (E) Plot showing relative (S+G2)/G1 ratio of RV proteins fused to CFP in MA104-Fucci at 24 hpt. Each RV protein fused to CFP was tested for functional expression by fluorescence microscopy at 24 hpt (data not shown). The relative (S+G2)/G1 ratio was calculated considering CFP only cells as value of 1. Data are represented as the mean ± SEM (t-test, *** p<0.001, n = 3). (F) Plot showing the percentage of the interphase stages of MA104-Fucci transfected with the indicated proteins fused to CFP. Data are represented as the mean ± SEM (t-test, ** p<0.01, *** p<0.001, n = 3).