To screen for SAFV protein(s) responsible for inducing apoptotic activity in HEp-2 and Vero cells, the vectors encoding the SAFV L, 1D, 2A, 2B, 2C, 3A, 3C, 3D, or BAX, or TMEV DA L protein, as well as empty vector, were initially transfected separately into HEp-2 cells or Vero cells to assay their expression profile. The result of the expression profile of respective Myc-tagged proteins by Western blot assay had been described (Xu et al. 2016). The expression levels of respective protein in Hep-2 cells and Vero cells transfected pXJ40-Myc-SAFV virus gene constructs, as well as pXJ40-Myc-DA L and pXJ40-MycBAX, at 24 h post-transfection were compared and shown in Table 1. In Table 1, the expression levels of respective proteins were comparable.
Table 1. Expression levels of respective protein in HEp-2 cells and Vero cells transfected pXJ40-Myc-SAFV virus gene constructs, as well as pXJ40-Myc-DA L and pXJ40-Myc-BAX, at 24 h post-transfectiona.
The individual transfected cells and cells treated with the chemical Staurosporine (STAU) were subsequently subjected to the cytotoxicity assay at 24 h post-transfection (Fig. 1). The result showed that cytotoxicity patterns in HEp-2 cells and Vero cells expressing individual viral protein were comparable. In Fig. 1, the cytotoxicity of cells expressing the 2B, 3C, BAX, and DA L proteins were significantly higher compared to the cells transfected with empty vector. This result suggests that the 2B and 3C proteins of SAFV are cytotoxic to HEp-2 and Vero cells, whereas the cytotoxicity assay of the L protein is comparatively low.
Figure 1. Cytotoxicity assay of HEp-2 cells (A) and Vero cells (B) expressing the SAFV L, 1D, 2A, 2B, 2C, 3A, 3C, 3D, TMEV DA L or BAX protein at 24 h post-transfection. STAU, cells treated with Staurosporine. EV, cells transfected with the empty vector pXJ40-Myc. -, cells without any treatment. The fluorometric readings of the cells transfected with empty vectors and each of other groups were compared by a paired Student's t test, and differences were considered significant at P < 0.01 (*). All experiments were repeated three times, and the average values with standard deviations are shown.
To determine the apoptotic activity of HEp-2 and Vero cells expressing SAFV L, 2B, or 3C protein, HEp-2 cells or Vero cells transfected with vectors encoding SAFV L, 2B, 3C, DA L, or BAX protein, as well as empty vector, were harvested at 24 h post-transfection for Western blot assay. The cells transfected with DA L protein and BAX protein served as positive controls of cells undergoing apoptosis, while empty vector served as negative control. As shown in Fig. 2, a greater proportion of the poly-ADP-ribose polymerase (PARP) proteins was cleaved in cells expressing DA L, SAFV 2B, 3C and BAX proteins, in contrast with limited cleavage of the PARP in cells transfected with vector encoding SAFV L protein or empty vector. The results obtained in HEp-2 and Vero cells were comparable. These results suggest that SAFV 2B and 3C proteins cause HEp-2 cells and Vero cells to undergo apoptosis, while its L protein is not responsible for inducing apoptotic cell death.
Figure 2. Western blot analysis of cleavage of poly-ADP-ribose polymerase (PARP) in HEp-2 and Vero cells expressing the L, DA L, 2B, 3C, or BAX protein at 24 h post-transfection. Membranes were probed with anti-PARP antibody. The bands represented at 89 kDa are the active form of PARP. MW, protein molecular mass in thousands Dalton (kDa). The actin staining was performed as protein loading control. The Western blot results shown here represent one of the three repeated experiments. EV, empty vector.
To confirm the apoptotic roles of SAFV L, 2B and 3C proteins, HEp-2 and Vero cells transfected with vectors encoding SAFV L, 2B, 3C, TMEV DA L or BAX protein, as well as empty vector, were fixed at 24 h and 48 h posttransfection and subjected to TUNEL assay. The apoptotic cells were labelled as the positive cells, and the percentage of the positive cells of the total transfected cells was calculated and analysed (Fig. 3). In both HEp-2 and Vero cells, the percentage of positive cells of the total transfected cells expressing SAFV 2B, 3C, TMEV DA L or BAX protein was significantly higher than that of cells transfected with empty vector at both 24 h and 48 h posttransfection. These findings confirm SAFV 2B and 3C proteins are proapoptotic, and its L protein does not have proapoptotic activity.
Figure 3. TUNEL assay of HEp-2 cells (A, B) and Vero cells (A, C) expressing SAFV L, 2B, 3C, TMEV DA L and BAX proteins. A TUNAL stained cells of HEp-2 and Vero cells expressing respective viral protein at 24 h post-transfection. Cell nuclei were stained with Hoechst 33258. Cells were observed with a fluorescence microscope (Leica SP8 laser scanning confocal microscope with a 63 ×/1.40 NA oil objective). Scale bar = 10 μm. B TUNAL assay of HEp-2 cells expressing respective viral protein at 24 h and 48 h posttransfection. C TUNAL assay of Vero cells expressing respective viral protein at 24 h and 48 h post-transfection. A thousand cells from at least 5 different optical fields were assessed in each experiment and the ratio of positive cells relative to total cells counted was scored. The similarities between the percentage of positive cells of the cells transfected with the pXJ40-Myc (empty vector) and that of each of other groups were compared with a paired Student's t-test, and P < 0.01 (*) was considered as significant. Results are representative of three independent experiments performed.
Taking into consideration that the 3C protein of SAFV was degraded by the cellular ubiquitin/26S proteasome after expression (Xu et al. 2016), we focused on the apoptotic activity of the 2B protein. SAFV 2B protein contains a transmembrane domain located between amino acid 78 and 96 of the sequence (Fig. 4A). In order to determine the function of the transmembrane domain, a plasmid encoding 2B△78-96 protein was constructed and transfected into HEp-2 and Vero cells in comparison with the wild-type 2B protein and empty vector. The cell lysates were collected at 24 h post-transfection, followed by incubation at 65 ℃ for 15 min (to reserve possible polymerization of the protein) before subjecting to Western blots analysis (Fig. 4B). The membranes were probed with anti-Myc antibody to show the expression of both 2B and 2B△78-96 proteins in their respective expected sizes, where the wild-type 2B protein lane showed four bands in one to four times of the expected size of the wild-type 2B protein. The membranes were probed with anti-PARP showing the cleavage of the PARP only in wild-type 2B protein, but not in 2B△78-96 protein. These results revealed that both 2B and 2B△78-96 proteins were expressed in HEp-2 and Vero cells, but only wildtype 2B protein formed tetramer in the transfected cells and induced the cells to undergo apoptosis.
Figure 4. Western blot analysis of the 2B and 2B△78-96 protein in HEp-2 and Vero cells at 24 h post-transfection. A Alignment of the amino acid sequences of SAFV 2B and 2B△78-96 proteins. The transmembrane domain of the 2B protein is highlighted in red. B Western blots analysis of the 2B and 2B△78-96 proteins in HEp-2 and Vero cells. Membranes were stained with anti-Myc or antiPARP antibody. The bands represent at 15 kDa is the monomer of the 2B protein, and 89 kDa is the active form of PARP. MW, protein molecular mass (in thousands Dalton, kDa). The actin staining was the protein loading control. The Western blot results shown here represent one of the three repeated experiments.