Mechanism Of Apoptosis In HeLa Cells Induced By The Hemagglutinin Protein Of Measles Virus

Measles virus (MV) encodes two envelope glycoproteins, haemagglutinin (H) and fusion (F) proteins, which are required for entry of the virus into cells. MV infection is initiated by the attachment of MV-H protein to its host cell receptors such as CD46or SLAM. It was demonstrated that the envelope glycoproteins of measles virus plays an important role in the immunosuppression of the patient. However, whether MV glycoproteins could induce cellular apoptosis was still of controversy, which remains to be further elucidated. In this study, the pro-apoptotic properties of MV glycoproteins were investigated in vitro. Plasmids pcDNA3.1-h for expressing MV-H and pHM6-f for expressing MV-F were transiently transfected or co-transfected into HeLa cells, the morphological, biochemical characteristics of cells in each group were then examined. After transfection, cells transfected with pcDNA3.1-h alone or pcDNA3.1-h/pHM6-f together appeared to undergo morphologic changes characteristic with apoptosis (i.e., cell rounding, shrinkage and detachment); cells transfected with pHM6-f alone or mock controls did not display these changes. MTT assay revealed that MV-H expression alone or its co-expression with MV-F significantly reduced the viability of HeLa cells, while MV-F expression alone did not. Hoechst33258staining analysis revealed that HeLa cells transfected with pcDNA3.1-h alone or pcDNA3.1-h/pHM6-f together showed condensed bright nuclei typical of apoptotic dead cells, whereas apoptotic nuclei was not observed in cells transfected with pHM6-f alone and mock controls. Characteristic DNA ladders were detected in cells transfected with pcDNA3.1-h alone or pcDNA3.1-h/pHM6-f together, but not in cells transfected with pHM6-f alone or in mock controls. The percentage of apoptotic cells (Annexin-V positive cells) in wells transfected with pcDNA3.1-h or pcDNA3.1-h+pHM6-f together were significantly increased when compared to percentages in mock controls, while the apoptotic percentage in pHM6-f transfected cells was similar to that in mock controls. Furthermore, the viability of cells or the percentage of apoptotic cells in pcDNA3.1-h-transfected was respectively similar to that in pcDNA3.1-h/pHM6-f co-transfected cell. These results demonstrated that expression of MV-H alone was sufficient to induce apoptosis of HeLa cells while MV-F was not; furthermore, MV-F had no effect on MV-H-induced apoptosis.We then explored the molecular mechanism responsible for the apoptosis induced by MV-H. We observed that the expression of both TRAIL and DR5in cells transfected with pcDNA3.1-h alone or pcDNA3.1-h+pHM6-f together was significantly up-regulated when compared to cells transfected with pHM6-f alone or un-transfected control. The expression levels of DR4, Fas and FasL remained the same among all experimental groups. We also found that treatment with the TRAIL-R2:Fc chimera, but not the anti-Fas antibody ZB4, significantly inhibited MV-H-mediated apoptosis. These data indicated that MV-H-induced apoptosis was associated with the extrinsic TRAIL signaling pathway.Since the intrinsic signaling pathway is always involved in apoptosis induced by viral proteins, we then analyzed the critical signaling events (such as the loss of△Ψm, the release of pro-apoptosis factor) in mitochondria. We discovered that the loss of mitochondrial trans-membrane potential (△Ψm), the release of pro-apoptotic proteins (Cyto c and AIF), and the increase of Bax expression and translation of Bax were observed in cells transfected with either pcDNA3.1-h alone or pcDNA3.1-h+pHM6-f together, but not in cells transfected with pHM6-f alone or mock controls. These results confirmed that modulation of the intrinsic mitochondrial pathway was also involved in MV-H-induced apoptosis.Furthermore, we also found that procaspase-8,-9and-3was respectively cleaved and activated by M-H. Apoptosis induced by MV-H was almost completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and the caspase-3inhibitor Z-DEVD-fmk; only partial inhibition was achieved by the caspase-8inhibitor Z-IETD-fmk and the caspase-9inhibitor Z-LEHD-fmk. These results suggested that MV-H-induced aopoptosis is caspase-dependent. In addition, we also found that MV-H induced the cleavage of Bid, and caspase-8inhibitor completely inhibited the cleavage of Bid, and partially suppressed caspase-9and-3activities. These data indicated that cross-talk between the extrinsic and intrinsic pathways occurred during MV-H-mediated apoptosis.The above data indicated that MV-H-induced apoptosis was a real sophisticated process. MV-H determined the fortune of cells through the apoptosis-related molecules involved in both cell death receptor-mediated extrinsic pathway and the mitochondria-controlled intrinsic pathway.Previously, it was demonstrated that the interaction of measles virus envelope glycoproteins and their viral receptor was involved in MV-mediated apoptosis, and contributed to MV-induced cells proliferation suppression. To further delineate the mechanism of apoptosis induced by MV-H, we determined whether the interaction of H and its cellular receptor CD46was responsible for MV-H-induced apoptosis. We found that remarked apoptosis occurrence, up-regulation of Trail/DR5expression was observed in H-positive (H-expressing) and H-negative cells, respectively. The apoptosis induced by MV-H was almost completely inhibited by anti-CD46and MV-H Ab. MV-H-induced up-regulation of Trail/DR5expression, loss of△Ψm and activation of caspases were also completely suppressed by anti-CD46and MV-H Ab, respectively. These results suggested that MV-H induced apoptosis through its interaction with cellular receptor CD46.