Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells
Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.
Keywords: HSV-1, Caspase-9, Cytochrome c, Bax
Apoptosis. 2007 Jan; 12(1): 19–35.
Published online 2006 Oct 31. doi: [10.1007/s10495-006-0330-3]
Martine Aubert,1,2 Lisa E. Pomeranz,1,3 and John A. Blahocorresponding author1
Author information Copyright and License information Disclaimer
1Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, NY 10029-6574 USA
2Program in Infectious Diseases, Fred Hutchinson CRC, Seattle, WA USA
3Laboratory of Molecular Genetics, The Rockefeller University, New York, NY USA
John A. Blaho, Email: firstname.lastname@example.org.
corresponding authorCorresponding author.
Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2799008/
Myxoma Virus M11L Blocks Apoptosis through Inhibition of Conformational Activation of Bax at the Mitochondria
Many viruses inhibit or retard apoptosis, a strategy that subverts one of the most ancient antiviral mechanisms. M11L, a myxoma virus-encoded antiapoptotic protein, has been previously shown to localize to mitochondria and block apoptosis of virus-infected cells (H. Everett, M. Barry, S. F. Lee, X. J. Sun, K. Graham, J. Stone, R. C. Bleackley, and G. McFadden, J. Exp. Med. 191:1487-1498, 2000; H. Everett, M. Barry, X. Sun, S. F. Lee, C. Frantz, L. G. Berthiaume, G. McFadden, and R. C. Bleackley, J. Exp. Med. 196:1127-1139, 2002; and G. Wang, J. W. Barrett, S. H. Nazarian, H. Everett, X. Gao, C. Bleackley, K. Colwill, M. F. Moran, and G. McFadden, J. Virol. 78:7097-7111, 2004). This protection from apoptosis involves constitutive-forming inhibitory complexes with the peripheral benzodiazepine receptor and Bak on the outer mitochondrial membrane. Here, we extend the study to investigate the interference of M11L with Bax activation during the process of apoptosis. Myxoma virus infection triggers an early apoptotic signal that induces rapid Bax translocation from cytoplasm to mitochondria, despite the existence of various viral antiapoptotic proteins. However, in the presence of M11L, the structural activation of Bax at the mitochondrial membrane, which is characterized by the occurrence of a Bax conformational change, is blocked in both M11L-expressing myxoma-infected cells and M11L-transfected cells under apoptotic stimulation. In addition, inducible binding of M11L to the mitochondrially localized Bax is detected in myxoma virus-infected cells and in M11L/Bax-cotransfected cells as measured by immunoprecipitation and tandem affinity purification analysis, respectively. Importantly, this inducible Bax/M11L interaction is independent of Bak, demonstrated by the complete block of Bax-mediated apoptosis in myxoma-infected cells that lack Bak expression. Our findings reveal that myxoma M11L modulates apoptosis by multiple independent strategies which all contribute to the blockade of apoptosis at the mitochondrial checkpoint.
Jin Su, Gen Wang, John W. Barrett, Timothy S. Irvine, Xiujuan Gao, Grant McFadden
Department of Microbiology and Immunology, University of Western Ontario and Robarts Research Institute, London, Ontario, Canada
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Myxoma Virus M11L Blocks Apoptosis through Inhibition of Conformational Activation of Bax at the Mitochondria | Journal of Virology https://jvi.asm.org/content/80/3/1140.short
The Rubella Virus Capsid Is an Anti-Apoptotic Protein that Attenuates the Pore-Forming Ability of Bax
Carolina S. Ilkow, Ing Swie Goping, Tom C. Hobman
Published: February 17, 2011https://doi.org/10.1371/journal.ppat.1001291
Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways, it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly, data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so, this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally, it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.
Among the variety of defense systems employed by mammalian cells to combat virus infection, apoptosis or programmed cell death is the most drastic response. Some large DNA viruses encode proteins whose sole function is to block apoptosis. Conversely, very little is known about whether RNA viruses encode analogous proteins. In many cases, RNA viruses are able to replicate before cell death occurs, which may be one reason why so little thought has been given to this topic. However, a number of RNA viruses, some of which are important human pathogens, have slow replication cycles and it stands to reason that they must block apoptosis during this time period. Here we show that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. Data from reverse genetic experiments suggest that the anti-apoptotic function of a virus-encoded protein is important for replication of an RNA virus. We anticipate that other slowly replicating RNA viruses may employ similar mechanisms and, as such, these studies have implications for development of novel anti-virals and vaccines.
Apoptosis is a common defense mechanism used by host cells to limit the spread of viral infections and consequently, a number of viruses have developed mechanisms to disrupt programmed cell death pathways. With few exceptions, all known viral apoptosis inhibitors are accessory proteins that are encoded by DNA viruses and therefore, a great deal of effort has focused on these proteins (reviewed in ). Ironically, even though RNA viruses cause the vast majority of viral diseases in humans, comparatively little is known about if or how these types of viruses interfere with apoptotic signaling. Among the exceptions are picornaviruses, a number of which encode “security” proteins (leader protein and 2BC) that can block apoptosis , . The mechanisms by which these proteins block apoptosis are not known and interestingly, caspase activation can still occur normally. These proteins may not actually prevent cell death per se, but rather, shift the balance toward necrotic cell death as opposed to apoptosis.
Hepatitis C virus (HCV) is known to modulate apoptotic signaling but unlike picornaviruses, this virus is not cytolytic and readily establishes persistent infections in vivo. HCV encodes a number of proteins that reportedly exhibit anti-apoptotic activity. For example, the nonstructural proteins NS2 and NS5A interfere with programmed cell death by different mechanisms , . The functions of HCV structural proteins in apoptotic signaling events are less clear; in particular, the core/capsid protein. The majority of data suggest that this protein acts to induce apoptosis although a number of published studies suggest otherwise (reviewed in ). Similarly, with one exception , expression of HCV E2 glycoprotein reportedly acts in a pro-apoptotic manner , , . By and large, these studies involved plasmid-based expression of individual HCV proteins and indeed the data provide much to ponder with respect how this virus interfaces with apoptotic pathways. However, it is still not clear how individual HCV proteins or those of any other RNA virus affect cell death during infection.
Multiple laboratories have reported that RV infection induces programmed cell death in a variety of cultured cell lines , , , ,  but it is worth noting that in virtually all cases, maximum synthesis of viral macromolecules and release of virions occur well before extensive apoptosis is observed. For example, in Vero cells, robust expression of structural proteins is first detected at 16 hours post-infection and secretion of infectious virions peaks 32 hours later . Conversely, late apoptotic events such as DNA fragmentation and expression of pro-apoptotic proteins p53 and p21 does not peak until 5–7 days post-infection . This indicates that that the majority of programmed cell death occurs long after the peak of virus production. Consistent with these observations, we show that RV infected cells are in fact, resistant to apoptosis for at least 48 hours post-infection.
Here, we provide evidence that in addition to functioning in virus assembly, the RV capsid protein is a potent inhibitor of apoptosis. With the possible exception of HCV capsid and E2, structural proteins of RNA viruses have been found to cause apoptosis rather than prevent cell death (reviewed in , , ). As far as we are aware, this is the first example of a structural protein from an RNA virus that functions to block cell death pathways through interactions with Bax. Mapping studies suggest that expression of the virus nonstructural proteins is the cause of RV-induced cell death , . Accordingly, counteracting apoptotic pathways that become activated by expression of these early proteins may be essential for efficient replication; a theory that is supported by data from reverse genetic experiments with the CR5A mutant.
The Rubella Virus Capsid Is an Anti-Apoptotic Protein that Attenuates the Pore-Forming Ability of Bax https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1001291