Figure 1.
BEV induced CPE in E. Derm cells.
E. Derm cells were mock-infected (A and D) or infected with BEV (5 pfu/cell), observed by phase-contrast microscopy and photographed at 24 (B and E) and 48 (C and F) hpi. Panels D, E and F represent enlarged areas (3×) of panels A, B and C respectively. (Scale bars, 200 µm).
Figure 2.
Electron microscopy of BEV infected E. Derm cells.
E. Derm cells were mock-infected (A) or infected with BEV (5 pfu/cell) (B), harvested at 24 hpi, fixed and embedded in Epoxi resin. Ultrathin sections of 60–70 nm were stained with lead citrate and uranyl acetate and observed by electron microscopy. Characteristic signs of apoptosis, such as chromatin condensation, membrane blebbing and cell disassembly, observed in the cell of panel B, were evident in some cells infected with BEV at 24 hpi. (Scale bars, 2 µm).
Figure 3.
Inhibition of protein synthesis in BEV infected E. Derm and MRC-5 cells.
E. Derm and MRC-5 cells were mock-infected (M) or infected with BEV (5 pfu/cell), and metabolically labeled with [35S]Met-Cys for 30 min at the time postinfection indicated on the top of the figures. Cellular extracts were analyzed by SDS-PAGE (12% polyacrylamide), and proteins were visualized after staining of the gels with coomassie blue (B and E), and labeled proteins were detected by autoradiography of the dried gel (A and D). The two major proteins that arise throughout the infection with BEV, identified as the structural M and N proteins, are indicated with arrows at the right side of each figure. A high molecular weight protein band (denoted by a solid arrowhead) and two other faint bands (denoted by empty arrowheads) likely representing the S precursor and cleaved products respectively, were also observed in [35S]Met-Cys-labeled extracts from E. Derm cells. The positions of the molecular mass markers in kDa are represented on the left. (C and F) Western blot analysis of E. Derm and MRC-5 cells infected with BEV (5 pfu/cell), collected at the indicated hpi and reacted with specific antibodies against the BEV structural S, M and N proteins (indicated at the right of the corresponding panel). The S precursor (denoted by a solid arrowhead), the 100 kDa cleaved product (denoted by an empty arrowhead), and the position of a protein recognized unespecifically by the anti-S monoclonal antibodies (denoted by an arrow) are indicated in the upper panels.
Figure 4.
Analysis of factors likely responsible for protein synthesis inhibition.
(A) For the analysis of eIF2α and PKR phosphorylation, E. Derm and MRC-5 cells were infected with BEV (5 pfu/cell), collected at different hpi, as indicated on the top of the figure, and subjected to Western blot analysis with antibodies against phosphorylated (α-eIF2α-P) and total (α-eIF2α-T) eIF2α, and phosphorylated (α-PKR-P) and total (α-PKR-T) PKR. The positions of molecular mass markers in kDa are indicated at the left of each panel. (B) For the rRNA degradation analysis, E. Derm and MRC-5 cells were infected with BEV (5 pfu/cell) and collected at the hpi indicated on the top of the figure. RNA from the infected and mock-infected (M) cell cultures was purified and the amount of RNA corresponding to 2×105 cells was fractionated in each lane of a 1% agarose gels stained with EtBr. The positions of the 28S and 18S rRNA molecules are indicated at the left of the figure.
Figure 5.
Cleavage of PARP and eIF4GI in BEV infected cells.
(A) E. Derm and MRC-5 cells were infected with BEV (5 pfu/cell) and collected at different hpi, as indicated on the top of the figure. As a negative control we used mock-infected cells (M). (B) E. Derm and MRC-5 cells were inoculated with BEV (5 pfu/cell) or UV-inactivated BEV (equivalent amount to 5 pfu/cell) and harvested at 24 (E. Derm) and 38 (MRC5) hpi. (A and B) Cell extracts were fractionated by SDS-PAGE (10% polyacrylamide) and analyzed by Western blot with anti-PARP and anti-eIF4GI antibodies. PARP and eIF4GI full-length proteins are indicated by solid arrowheads and their degradation products are indicated by empty arrowheads. The positions of molecular mass markers in kDa are indicated at the left of each panel.
Figure 6.
DNA degradation in BEV infected cells.
(A) E. Derm cells were infected with BEV (5 pfu/cell) and a nuclei staining kinetic study was performed. As a control, mock-infected cells were used (Mock). Cells were fixed with PFA at the indicated hpi, and nuclei were stained with Hoechst. (B and C) Quantification of DNA degradation in BEV infected cells by cell cycle analysis with flow cytometry. E. Derm and MRC-5 cells infected with BEV (5 pfu/cell) were fixed and PI stained at the indicated hpi. The percentages of apoptotic cells were determined as the subG0-G1 fraction. (B) Cellular cycle graphs obtained in a representative experiment. (C) Graphic representation of the percentages of apoptotic BEV infected cells at different hpi. Data represent the media ± the standard deviation of three experiments.
Figure 7.
Analysis of the effect of caspase inhibitors z-VAD-fmk, z-IETD-fmk and z-LEHD-fmk on BEV infected cells.
E. Derm and MRC-5 cells were infected with BEV (5 pfu/cell) and treated with the general caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (z-LEHD-fmk) (50 µM each). Infected but untreated cells were used as a control (A, B and C). Cells were collected at 48 hpi, and the cellular DNA content was analyzed by flow cytometry after PI staining. (A) Cell cycle graph obtained in a representative experiment. (B) Graphic representation of the percentage of apoptotic cells ± the standard deviation from two experiments. Asterisks indicate statistically significant differences between the means relative to the corresponding control (**p<0.05, *p<0.1; Student's t test). (C) E. Derm cells collected at 24 hpi, and MRC-5 cells collected at 38 hpi were analyzed by Western blot with anti-eIF4GI antibody, and anti-BEV-N and anti-actin antibodies as infection and load control respectively. The position of the cleaved eIF4GI protein is indicated at the left of the panel. (D) E. Derm and MRC-5 cells were infected with BEV at 5 pfu/cell in the absence or presence of z-VAD-fmk, and the supernatants were collected at 8, 16, 24, 32 and 48 hpi and subjected to virus titration. Graphic representation of the media ± the standard deviation of three independent experiments. There were no statistically significant differences between the values in the absence and presence of z-VAD-fmk for any of the two cell lines and at any of the time points tested (p>0.5; Student's t test).
Figure 8.
Cytochrome c release from the mitochondria and Bid cleavage upon BEV infection.
E. Derm and MRC-5 cells were mock-infected or infected with BEV (5 pfu/cell), harvested at 24 hpi, and fractionated into cytosolic and membrane-bound organellar (mitochondrial) fractions. Cells treated with staurosporine for 6 h, included as a positive control for mitochondrial pathway activation, were also fractionated. Cytosolic and mitochondrial fractions from mock-infected cells (lanes 2), cells treated with staurosporine (lanes 1) or cells infected for 24 h (lanes 3) were analyzed by Western blot with antibodies against cytochrome c and Bid proteins. The positions of the Bid protein and the truncated Bid (tBid) are indicated at the left of the panel B. Reactivity with anti-actin antibody was used as a load control.