Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.
The RNA alphavirus Semliki Forest (SFV) triggers apoptosis in various mammalian cells, but it has remained controversial at what infection stage and by which signalling pathways host cells are killed. Both RNA synthesis-dependent and -independent initiation processes and mitochondrial as well as death receptor signalling pathways have been implicated. Here, we show that SFV-induced apoptosis is initiated at the level of RNA replication or thereafter. Moreover, by expressing antiapoptotic genes from recombinant SFV (replicons) and by using neutralizing reagents and gene-knockout cells, we provide clear evidence that SFV does not require CD95L-, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-or tumor necrosis factormediated signalling but mitochondrial Bak to trigger cytochrome c release, the fall in the mitochondrial membrane potential, apoptotic protease-activating factor-1/caspase-9 apoptosome formation and caspase-3/-7 activation. Of seven BH3-only proteins tested, only Bid contributed to effective SFV-induced apoptosis. However, caspase-8 activation and Bid cleavage occurred downstream of Bax/Bak, indicating that truncated Bid formation serves to amplify rather than trigger SFV-induced apoptosis. Our data show that SFV sequentially activates a mitochondrial, Bak-mediated, caspase-8-dependent and Bid-mediated death signalling pathway that can be accurately dissected with gene-knockout cells and SFV replicons carrying antiapoptotic genes.
A great variety of viruses have been engineered to serve as expression vectors. Among them, the alphaviruses Semliki Forest virus and Sindbis virus represent promising tools for heterologous gene expression in a wide variety of host cells. Several applications have already been described in neurobiological studies, in gene therapy, for vaccine development and in cancer therapy. Both viruses trigger stress pathways in the cells they infect, sometimes culminating in the death of the host. This inherent property is either an advantage or a drawback, depending on the type of application.This review covers the development and applications of alphavirus vectors and, as our work has been mainly with Semliki Forest virus, we have focused on this virus with special emphasis on how the understanding of Semliki Forest virus cytotoxicity enables it to be manipulated and used. Biology of alphavirusesGenomic organization. The single-stranded, around 12 kb long genomes of SFV and SIN are divided into two open reading frames (ORF). The first ORF encodes four non-structural proteins, designated nsP1 to nsP4, responsible for transcription and replication of viral RNA. The second ORF, under the control of a 26S subgenomic promoter, codes for the structural proteins required for the encapsidation of the viral genome and the proper assembly into enveloped particles. They include the capsid protein, the glycoproteins E1, E2 and E3 and the 6K protein. The structural proteins are not necessary for viral replication, but are required for virus propagation, together with the packaging signal located in the coding region of nsP2 in SFV and of nsP1 in SIN (Strauss & Strauss, 1994).Life cycle. After attachment of the virion to a cell receptor via the exposed E2 portion of the spikes, the virion enters endosomes. The release of the nucleocapsid into the cytoplasm occurs upon fusion of the viral and endosomal membranes as a consequence of conformational changes in the glycoprotein spikes induced by the endosomal acidic environment. After dissolution of the nucleocapsid, the liberated genomic RNA codes for the polyprotein of non-structural proteins. Its sequential processing by the cysteine protease activity of nsP2 determines the specificity of the replication complex. Initially, the primary complex synthesizes the negative-sense RNA, using the original genomic RNA as a template. Later, upon assembly of a complex composed of the four individual proteins, C The Physiological Society 2004
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