The SARS-Coronavirus Membrane protein induces apoptosis through modulating the Akt survival pathway

Abstract

A number of viral gene products are capable of triggering apoptotic cell death through interfering with cellular signaling cascades, including the Akt kinase pathway. In this study, the pro-apoptotic role of the SARS-CoV Membrane (M) structural protein is described. We found that the SARS-CoV M protein induced apoptosis in both HEK293T cells and transgenic Drosophila. We further showed that M protein-induced apoptosis involved mitochondrial release of cytochrome c protein, and could be suppressed by caspase inhibitors. Over-expression of M caused a dominant rough-eye phenotype in adult Drosophila. By performing a forward genetic modifier screen, we identified phosphoinositide-dependent kinase-1 (PDK-1) as a dominant suppressor of M-induced apoptotic cell death. Both PDK-1 and Akt kinases play essential roles in the cell survival signaling pathway. Altogether, our data show that SARS-CoV M protein induces apoptosis through the modulation of the cellular Akt pro-survival pathway and mitochondrial cytochrome c release.

Fig. 1

Expression and subcellular localization of SARS-CoV Membrane protein in HEK293T cells. (A) mRNA expression level of the SARS-CoV M gene was examined by RT-PCR. Cells transfected with pcDNA3.1(+)-Membrane plasmid showed M mRNA expression at 48 h post-transfection, while no expression was detected in the untransfected and empty vector controls. GAPDH was used as loading control. RT(+) and RT(−) represent the presence and absence of reverse transcriptase enzyme in the RT reaction, respectively. (B–G) Subcellular localization of M protein was determined by immunofluorescence staining. The M protein displayed a punctate cytoplasmic staining pattern (E, in green) and partially localized (G) with the Golgi body (F, in red). Untransfected cells showed no expression of Membrane protein (B). Scale bars represent 16 μm.

Fig. 2

Induction of apoptosis by over-expression of M protein in HEK293T cells. (A–L) M protein expression induced nuclear condensation in HEK293T cells. Expression of M protein was only detected in transfected cells (G, in green), but not in untransfected (A and J) and empty vector (D) controls. Hoechst 33342 was used to label cell nuclei (B, E, H and K, in blue). Cells expressed with M protein showed nuclear condensation (I, arrowheads) at 48 h post-transfection. Untransfected cells treated with 1 μM staurosporine for 8 h also displayed nuclear condensation (K, arrowheads), while untreated (B) and empty vector (E) controls showed normal nuclear morphology. (M) The percentage of cells showed nuclear condensation was quantified. Results were plotted as percentage of cells showed nuclear condensation and expressed as means + SEM of three independent experiments. At least 100 cells were counted in each experiment. Scale bars represent 16 μm. ^∗p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 3

Cytochrome c and caspases are involved in M-induced apoptosis. (A–F) M protein expression induced mis-localization of cytochrome c protein in HEK293T cells. When cells were either transfected with M (C) for 48 h or treated with 1 μM staurosporine for 8 h (E), mis-localization of cytochrome c protein was observed. Untransfected cells were used as control (A). Respective phase-contrast images are shown in B, D and F. Scale bars represent 16 μm. (G) M-induced nuclear condensation was inhibited by caspase inhibitors (at 50 μM) z-DQMD-fmk (caspase-3 inhibitor V), z-IETD-fmk (caspase-8 inhibitor II) and z-LEHD-fmk (caspase-9 inhibitor I). Results were plotted as percentage of cells showed nuclear condensation and expressed as means + SEM of three independent experiments. At least 100 cells were counted in each experiment. ^∗p < 0.05.

Fig. 4

Induction of rough-eye phenotype by over-expression of M protein in Drosophila. (A–C) Over-expression of the M protein in eye tissues resulted in rough-eye phenotype as characterized by loss of regularity of the adult external eye structure (C), whereas the gmr-GAL4 driver alone control (A) and over-expression of the EGFP protein (B) showed no dominant external eye phenotype. (D–F) Subcellular localization of M protein in the Drosophila third instar imaginal eye disc tissues. The M protein showed a distinct punctate cytoplasmic expression pattern (F, in green), whereas the expression EGFP control protein showed homogeneous intracellular staining (E, in green). Cell nuclei were stained by propidium iodide (in red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 5

Induction of apoptosis by over-expression of M protein in Drosophila. (A–E) gmr-GAL4 alone control showed no dominant eye phenotype (A). A rough-eye phenotype was observed upon M protein over-expression (B), and was suppressed by co-expression with anti-apoptotic genes DIAP1 (C), P35 (D), and apo-cytochrome cdc3^EP2305 (E). (F–J) M over-expression induced apoptosis in third instar imaginal eye discs as indicated by the increase in the number of acridine orange (AO)-stained cells (G). Induction of apoptosis by M protein was suppressed by the co-expression of anti-apoptotic genes DIAP1 (H), P35 (I), and apo-cytochrome cdc3^EP2305 (J). Arrows indicate morphogenetic furrows. (K) The number of AO-positive cells in each genotype was quantified. Results were plotted as the number of AO-positive cells and expressed as means + SEM of three-independent experiments. At least nine imaginal eye discs were analyzed in each genotype. ^∗p < 0.05 versus gmr-GAL4; ^#p < 0.05 versus gmr-GAL4 UAS-Membrane.

Fig. 6

Suppression of M-induced apoptosis by phosphoinositide-dependent kinase-1 over-expression in Drosophila. (A–C) PDK-1 over-expression suppressed M-induced rough-eye phenotype. gmr-GAL4 alone control showed no dominant eye phenotype (A). The M-induced rough-eye phenotype (B) was suppressed by over-expression of PDK-1 (C). In the presence of GAL4, PDK-1^EP837 causes over-expression of endogenous PDK-1. (D–F) PDK-1 over-expression suppressed M-induced apoptosis as shown by acridine orange (AO) staining. The elevated number of AO-positive cells caused by M protein over-expression in imaginal eye discs (E) was reduced upon the over-expression of PDK-1 (F). Arrows indicate morphogenetic furrows. (G) The number of AO-positive cells in each genotype was quantified. Results were plotted as the number of AO-positive cells and expressed as means + SEM of three-independent experiments. At least nine imaginal eye discs were analyzed in each genotype. ^∗p < 0.05 versus gmr-GAL4; ^#p < 0.05 versus gmr-GAL4 UAS-Membrane.

Fig. 7

Down-regulation of Akt protein phosphorylation in transgenic Drosophila over-expressing M protein. (A) Western blot analysis showed that M expression reduced Akt protein phosphorylation without altering total Akt protein level. β-tubulin was used as loading control. Phospho- (B) and total-Akt protein (C) levels were quantified and normalized against the β-tubulin loading control. Results were plotted as the percentage change of band intensity and expressed as means + SEM of five independent experiments. ^∗p < 0.05.