Microbes and Infection 16 (2014) 175e177 www.elsevier.com/locate/micinf
Highlight
What doesn’t kill flu, only makes flu stronger In the closing year of the First World War, a new threat loomed on the horizon. H1N1 pandemic influenza would soon make its deadly rampage across the world, ironically striking down mainly those in the prime of their lives, many of whom were returning from the trenches. The 1918e1919 unrighteously named ‘Spanish’ flu pandemic is thought to have led to the loss of 50 million lives [2], which incidentally is substantially higher than the number of people killed in the First World War itself. Nonetheless, the vast majority of those infected with influenza (>97%) in 1918 survived, in an age without influenza vaccines, antivirals, or antibiotics. Despite the full arsenal of 21st century medicine, the re-emergence in 2009 of the H1N1 subtype in the form of novel pandemic influenza A (aka Swine flu) nonetheless caused 19,000 deaths according to the World Health Organization (WHO). The flu season is upon us again and the predominant virus this year is the troublesome H1N1. Influenza A viruses are highly contagious pathogens for humans and several animal species. The control of these unruly pathogens will come not only from the development of effective vaccines, but also from an in depth understanding of the biology of these viruses and their interactions with the host organism. The genomes of influenza A viruses are small, and code for only 11 proteins. Due to this limited coding capacity, these viruses extensively manipulate the functions of the host cell to achieve efficient replication of their viral genome. For example, the NS1 protein interferes with the production of the cytokines interferon (IFN)-a and -b, which constitute a major part of the antiviral response. Indeed, it came as somewhat of a surprise when influenza viruses with deleted NS1 genes proved to be much stronger inducers of IFN-a/b than their wildtype counterparts [3,4]. The virus-mediated attenuation of host defence strategies has a clear logic for the survival and spread of the invading pathogen. However, recent data suggest that the virus may also hijack host defence mechanisms and turn them to their advantage. It is well established that infection with influenza induces apoptosis in cultured epithelial cells and leukocytes, as well as murine and human pulmonary cell populations in vivo [5e7]. Programmed cell death (apoptosis) has been traditionally viewed as a host cell defence mechanism to limit viral production. Two major apoptotic pathways have been defined: the ‘extrinsic’ pathway, which is mediated by the binding of ‘death ligands’ TNF-a, TRAIL, or FasL to transmembrane receptors of the TNFR superfamily, and the ‘intrinsic’ or ‘mitochondrial’
pathway which involves a diverse array of stimuli that activate pro-apoptotic members of the Bcl-2 family (Bax, Bak) to induce mitochondrial membrane permeabilization. Viralinduced death signals play a key role in directing and coordinating innate and adaptive immune responses. For example, TRAIL and FasL are expressed on the surface of influenza A infected cells in proportions that vary according to cell type [8]. FasL is an important factor that mediates the destruction of virus-infected cells by antigen specific T cells [9], and TRAIL may mediate cytotoxic T cell responses [10]. Logically therefore, many viruses interfere with components of apoptotic signalling to prevent this cellular response. Intriguingly, in addition to the reported anti-apoptotic functions of the multi-tasking Influenza A viral protein NS1 [11], this viral protein has also been reported to have apoptosis-inducing activities [12,13]. Furthermore, the viral PB1-F2 protein was originally described as a mitochondrial protein that can induce apoptosis in monocytic cells [14]. The existence of viral pro-apoptotic functions suggests that some benefit to the virus is reaped from apoptosis. In numerous cellular models, inhibiting apoptosis was shown to limit viral replication. The earliest of such studies showed that overexpression of the anti-apoptotic protein Bcl-2 was associated with a reduction in progeny virus titres [5,15]. Another landmark study showed that inhibition of the central components of the cell’s apoptotic machinery, cysteinyl proteases termed caspases, was also correlated with a reduction in influenza A virus propagation [16]. The 2009 strain of H1N1 novel pandemic influenza (H1N1pdm) activates apoptosis in human lung epithelial cells [17,18], although the apoptotic pathways involved and the consequences of apoptosis on viral propagation have not been defined. In this issue of Microbes and Infection, Hewlett et al. investigate the apoptotic pathways activated by H1N1pdm, and their effect on viral replication [1]. Infection of human lung epithelial cells with novel H1N1pdm virus led to the activation of components of both the extrinsic and intrinsic apoptotic pathways, including FasL, FADD, and p53, whereas the abundance of anti-apoptotic factors FLIP and Bcl-XL were reduced. The forced expression of pro-apoptotic factors (FasL, FADD, p53) in cells infected with H1N1pdm was associated with higher viral loads than the forced expression of a LacZ control gene. Conversely, the overexpression of anti-apoptotic factors (FLIP), or the chemical inhibition of caspase 3 or 8 was associated with lower viral loads than the control. These observations suggest
http://dx.doi.org/10.1016/j.micinf.2014.01.006 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Highlight / Microbes and Infection 16 (2014) 175e177
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that H1N1pdm takes advantage of the host cell apoptosis machinery and uses it for its own replication process. How then does death of the infected cell spell victory for its resident viral particles? The viral genome is replicated and transcribed inside the host nucleus, a feature that requires bidirectional transport through the nuclear membrane. Export of viral RNP complexes may be a rate limiting step in the formation of progeny virus particles [16]. The activation of caspases however leads to the concomitant widening of nuclear pores which may regulate RNP export by allowing the passage of large proteins [19]. All together, these data paint the worrying picture that killing the infected cell actually makes the virus more powerful. Targeted strategies to inhibit apoptosis would certainly be beneficial for the lungs, which can sustain massive tissue damage due to this phenomenon. Nonetheless, the discovery of both proand anti-apoptotic functions in various strains of influenza suggests that apoptosis may be beneficial for the virus only at certain stages of its life cycle. Anti-apoptotic signals may predominate in the early stages of the viral life cycle, during which the viral genome must first be multiplied, and pro-apoptotic signals may dominate in the late stages of infection when newly formed viral RNPs must be exported from the nucleus [20]. How the timing of these events is carefully coordinated will need to be worked out. They are also likely to vary depending on the virus. H1N1 may be the harbinger of winter flu occupying this month’s media headlines, but with the production of novel genotypes via reassortment following mixed infections, new strains and more pandemics are set to come.
In a Nutshell Infection of human lung epithelial cells with novel pandemic influenza A (H1N1pdm) virus led to the activation of components of extrinsic and intrinsic apoptotic pathways. Notably, H1N1pdm infection increased the abundance of p53. In human lung epithelial cells infected with H1N1pdm, forced expression of pro-apoptotic factors (FasL, FADD, p53) was associated with higher viral loads than the forced expression of a LacZ control gene. Conversely, the forced expression of anti-apoptotic factors (FLIP) was associated with lower viral loads than the control. Chemical inhibition of caspase 3, caspase 8, or FADD was also associated with lower viral loads than mock-treated cells. The phosphorylation of components of the MAPK signalling cascade, including p38, ERK, and JNK, were high in H1N1pdm-infected cells. Inhibition of these pathways was associated with lower viral loads than mocktreated cells.
Background Influenza A viruses are highly contagious pathogens for humans and animals. Novel pandemic influenza H1N1 virus killed an estimated 19,000 people in the 2009 pandemic and continues to be a major source of burden today. The development of effective anti-viral agents requires an understanding of the complex interactions between the virus and the host organism. Influenza viruses have small genomes and thus many viral gene products have co-evolved to extensively manipulate the host cell machinery. Apoptosis can be seen as a cellular defence mechanism to limit the spread of infection. Intriguingly however, both anti-apoptotic and proapoptotic functions have been described for influenza viruses. This suggests that in some circumstances the virus may benefit from apoptosis. H1N1 novel pandemic influenza activates apoptosis in human lung epithelial cells, although the apoptotic pathways involved and the consequences of apoptosis on viral propagation have not been defined.
Drawing by Sophia Hafner.
References [1] Wang X, Tan J, Zoueva O, Zhao J, Ye Z, et al. Novel pandemic influenza A (H1N1) virus infection modulates apoptotic pathways that impact its replication in A549 cells. Microbes Infect 2014;16:178e86. [2] Johnson NP, Mueller J. Updating the accounts: global mortality of the 1918e1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105e15. [3] Garcia-Sastre A, Egorov A, Matassov D, Brandt S, Levy DE, et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 1998;252:324e30.
Highlight / Microbes and Infection 16 (2014) 175e177 [4] Dauber B, Heins G, Wolff T. The influenza B virus nonstructural NS1 protein is essential for efficient viral growth and antagonizes beta interferon induction. J Virol 2004;78:1865e72. [5] Hinshaw VS, Olsen CW, Dybdahl-Sissoko N, Evans D. Apoptosis: a mechanism of cell killing by influenza A and B viruses. J Virol 1994;68:3667e73. [6] Fesq H, Bacher M, Nain M, Gemsa D. Programmed cell death (apoptosis) in human monocytes infected by influenza A virus. Immunobiology 1994;190:175e82. [7] Mori I, Komatsu T, Takeuchi K, Nakakuki K, Sudo M, et al. In vivo induction of apoptosis by influenza virus. J Gen Virol 1995;76(Pt 11):2869e73. [8] Herold S, Ludwig S, Pleschka S, Wolff T. Apoptosis signaling in influenza virus propagation, innate host defense, and lung injury. J Leukoc Biol 2012;92:75e82. [9] Topham DJ, Tripp RA, Doherty PC. CD8þ T cells clear influenza virus by perforin or Fas-dependent processes. J Immunol 1997;159:5197e200. [10] Brincks EL, Katewa A, Kucaba TA, Griffith TS, Legge KL. CD8 T cells utilize TRAIL to control influenza virus infection. J Immunol 2008;181:4918e25. [11] Zhirnov OP, Konakova TE, Wolff T, Klenk HD. NS1 protein of influenza A virus down-regulates apoptosis. J Virol 2002;76:1617e25. [12] Schultz-Cherry S, Dybdahl-Sissoko N, Neumann G, Kawaoka Y, Hinshaw VS. Influenza virus ns1 protein induces apoptosis in cultured cells. J Virol 2001;75:7875e81. [13] Lam WY, Tang JW, Yeung AC, Chiu LC, Sung JJ, et al. Avian influenza virus A/HK/483/97(H5N1) NS1 protein induces apoptosis in human airway epithelial cells. J Virol 2008;82:2741e51. [14] Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, et al. A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 2001;7:1306e12.
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[15] Olsen CW, Kehren JC, Dybdahl-Sissoko NR, Hinshaw VS. bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation. J Virol 1996;70:663e6. [16] Wurzer WJ, Planz O, Ehrhardt C, Giner M, Silberzahn T, et al. Caspase 3 activation is essential for efficient influenza virus propagation. EMBO J 2003;22:2717e28. [17] Yang P, Deng J, Li C, Zhang P, Xing L, et al. Characterization of the 2009 pandemic A/Beijing/501/2009 H1N1 influenza strain in human airway epithelial cells and ferrets. PLoS One 2012;7:e46184. [18] Yang N, Hong X, Yang P, Ju X, Wang Y, et al. The 2009 pandemic A/ Wenshan/01/2009 H1N1 induces apoptotic cell death in human airway epithelial cells. J Mol Cell Biol 2011;3:221e9. [19] Faleiro L, Lazebnik Y. Caspases disrupt the nuclear-cytoplasmic barrier. J Cell Biol 2000;151:951e9. [20] Zhirnov OP, Klenk HD. Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling. Apoptosis 2007;12:1419e32.
Dr. Emma Louise Walton Alex Edelman & Associates 24 rue Ampe`re Malakoff 92240 France E-mail address: [email protected] 23 January 2014 Available online 12 February 2014
Highlight
What doesn’t kill flu, only makes flu stronger In the closing year of the First World War, a new threat loomed on the horizon. H1N1 pandemic influenza would soon make its deadly rampage across the world, ironically striking down mainly those in the prime of their lives, many of whom were returning from the trenches. The 1918e1919 unrighteously named ‘Spanish’ flu pandemic is thought to have led to the loss of 50 million lives [2], which incidentally is substantially higher than the number of people killed in the First World War itself. Nonetheless, the vast majority of those infected with influenza (>97%) in 1918 survived, in an age without influenza vaccines, antivirals, or antibiotics. Despite the full arsenal of 21st century medicine, the re-emergence in 2009 of the H1N1 subtype in the form of novel pandemic influenza A (aka Swine flu) nonetheless caused 19,000 deaths according to the World Health Organization (WHO). The flu season is upon us again and the predominant virus this year is the troublesome H1N1. Influenza A viruses are highly contagious pathogens for humans and several animal species. The control of these unruly pathogens will come not only from the development of effective vaccines, but also from an in depth understanding of the biology of these viruses and their interactions with the host organism. The genomes of influenza A viruses are small, and code for only 11 proteins. Due to this limited coding capacity, these viruses extensively manipulate the functions of the host cell to achieve efficient replication of their viral genome. For example, the NS1 protein interferes with the production of the cytokines interferon (IFN)-a and -b, which constitute a major part of the antiviral response. Indeed, it came as somewhat of a surprise when influenza viruses with deleted NS1 genes proved to be much stronger inducers of IFN-a/b than their wildtype counterparts [3,4]. The virus-mediated attenuation of host defence strategies has a clear logic for the survival and spread of the invading pathogen. However, recent data suggest that the virus may also hijack host defence mechanisms and turn them to their advantage. It is well established that infection with influenza induces apoptosis in cultured epithelial cells and leukocytes, as well as murine and human pulmonary cell populations in vivo [5e7]. Programmed cell death (apoptosis) has been traditionally viewed as a host cell defence mechanism to limit viral production. Two major apoptotic pathways have been defined: the ‘extrinsic’ pathway, which is mediated by the binding of ‘death ligands’ TNF-a, TRAIL, or FasL to transmembrane receptors of the TNFR superfamily, and the ‘intrinsic’ or ‘mitochondrial’
pathway which involves a diverse array of stimuli that activate pro-apoptotic members of the Bcl-2 family (Bax, Bak) to induce mitochondrial membrane permeabilization. Viralinduced death signals play a key role in directing and coordinating innate and adaptive immune responses. For example, TRAIL and FasL are expressed on the surface of influenza A infected cells in proportions that vary according to cell type [8]. FasL is an important factor that mediates the destruction of virus-infected cells by antigen specific T cells [9], and TRAIL may mediate cytotoxic T cell responses [10]. Logically therefore, many viruses interfere with components of apoptotic signalling to prevent this cellular response. Intriguingly, in addition to the reported anti-apoptotic functions of the multi-tasking Influenza A viral protein NS1 [11], this viral protein has also been reported to have apoptosis-inducing activities [12,13]. Furthermore, the viral PB1-F2 protein was originally described as a mitochondrial protein that can induce apoptosis in monocytic cells [14]. The existence of viral pro-apoptotic functions suggests that some benefit to the virus is reaped from apoptosis. In numerous cellular models, inhibiting apoptosis was shown to limit viral replication. The earliest of such studies showed that overexpression of the anti-apoptotic protein Bcl-2 was associated with a reduction in progeny virus titres [5,15]. Another landmark study showed that inhibition of the central components of the cell’s apoptotic machinery, cysteinyl proteases termed caspases, was also correlated with a reduction in influenza A virus propagation [16]. The 2009 strain of H1N1 novel pandemic influenza (H1N1pdm) activates apoptosis in human lung epithelial cells [17,18], although the apoptotic pathways involved and the consequences of apoptosis on viral propagation have not been defined. In this issue of Microbes and Infection, Hewlett et al. investigate the apoptotic pathways activated by H1N1pdm, and their effect on viral replication [1]. Infection of human lung epithelial cells with novel H1N1pdm virus led to the activation of components of both the extrinsic and intrinsic apoptotic pathways, including FasL, FADD, and p53, whereas the abundance of anti-apoptotic factors FLIP and Bcl-XL were reduced. The forced expression of pro-apoptotic factors (FasL, FADD, p53) in cells infected with H1N1pdm was associated with higher viral loads than the forced expression of a LacZ control gene. Conversely, the overexpression of anti-apoptotic factors (FLIP), or the chemical inhibition of caspase 3 or 8 was associated with lower viral loads than the control. These observations suggest
http://dx.doi.org/10.1016/j.micinf.2014.01.006 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Highlight / Microbes and Infection 16 (2014) 175e177
176
that H1N1pdm takes advantage of the host cell apoptosis machinery and uses it for its own replication process. How then does death of the infected cell spell victory for its resident viral particles? The viral genome is replicated and transcribed inside the host nucleus, a feature that requires bidirectional transport through the nuclear membrane. Export of viral RNP complexes may be a rate limiting step in the formation of progeny virus particles [16]. The activation of caspases however leads to the concomitant widening of nuclear pores which may regulate RNP export by allowing the passage of large proteins [19]. All together, these data paint the worrying picture that killing the infected cell actually makes the virus more powerful. Targeted strategies to inhibit apoptosis would certainly be beneficial for the lungs, which can sustain massive tissue damage due to this phenomenon. Nonetheless, the discovery of both proand anti-apoptotic functions in various strains of influenza suggests that apoptosis may be beneficial for the virus only at certain stages of its life cycle. Anti-apoptotic signals may predominate in the early stages of the viral life cycle, during which the viral genome must first be multiplied, and pro-apoptotic signals may dominate in the late stages of infection when newly formed viral RNPs must be exported from the nucleus [20]. How the timing of these events is carefully coordinated will need to be worked out. They are also likely to vary depending on the virus. H1N1 may be the harbinger of winter flu occupying this month’s media headlines, but with the production of novel genotypes via reassortment following mixed infections, new strains and more pandemics are set to come.
In a Nutshell Infection of human lung epithelial cells with novel pandemic influenza A (H1N1pdm) virus led to the activation of components of extrinsic and intrinsic apoptotic pathways. Notably, H1N1pdm infection increased the abundance of p53. In human lung epithelial cells infected with H1N1pdm, forced expression of pro-apoptotic factors (FasL, FADD, p53) was associated with higher viral loads than the forced expression of a LacZ control gene. Conversely, the forced expression of anti-apoptotic factors (FLIP) was associated with lower viral loads than the control. Chemical inhibition of caspase 3, caspase 8, or FADD was also associated with lower viral loads than mock-treated cells. The phosphorylation of components of the MAPK signalling cascade, including p38, ERK, and JNK, were high in H1N1pdm-infected cells. Inhibition of these pathways was associated with lower viral loads than mocktreated cells.
Background Influenza A viruses are highly contagious pathogens for humans and animals. Novel pandemic influenza H1N1 virus killed an estimated 19,000 people in the 2009 pandemic and continues to be a major source of burden today. The development of effective anti-viral agents requires an understanding of the complex interactions between the virus and the host organism. Influenza viruses have small genomes and thus many viral gene products have co-evolved to extensively manipulate the host cell machinery. Apoptosis can be seen as a cellular defence mechanism to limit the spread of infection. Intriguingly however, both anti-apoptotic and proapoptotic functions have been described for influenza viruses. This suggests that in some circumstances the virus may benefit from apoptosis. H1N1 novel pandemic influenza activates apoptosis in human lung epithelial cells, although the apoptotic pathways involved and the consequences of apoptosis on viral propagation have not been defined.
Drawing by Sophia Hafner.
References [1] Wang X, Tan J, Zoueva O, Zhao J, Ye Z, et al. Novel pandemic influenza A (H1N1) virus infection modulates apoptotic pathways that impact its replication in A549 cells. Microbes Infect 2014;16:178e86. [2] Johnson NP, Mueller J. Updating the accounts: global mortality of the 1918e1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105e15. [3] Garcia-Sastre A, Egorov A, Matassov D, Brandt S, Levy DE, et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 1998;252:324e30.
Highlight / Microbes and Infection 16 (2014) 175e177 [4] Dauber B, Heins G, Wolff T. The influenza B virus nonstructural NS1 protein is essential for efficient viral growth and antagonizes beta interferon induction. J Virol 2004;78:1865e72. [5] Hinshaw VS, Olsen CW, Dybdahl-Sissoko N, Evans D. Apoptosis: a mechanism of cell killing by influenza A and B viruses. J Virol 1994;68:3667e73. [6] Fesq H, Bacher M, Nain M, Gemsa D. Programmed cell death (apoptosis) in human monocytes infected by influenza A virus. Immunobiology 1994;190:175e82. [7] Mori I, Komatsu T, Takeuchi K, Nakakuki K, Sudo M, et al. In vivo induction of apoptosis by influenza virus. J Gen Virol 1995;76(Pt 11):2869e73. [8] Herold S, Ludwig S, Pleschka S, Wolff T. Apoptosis signaling in influenza virus propagation, innate host defense, and lung injury. J Leukoc Biol 2012;92:75e82. [9] Topham DJ, Tripp RA, Doherty PC. CD8þ T cells clear influenza virus by perforin or Fas-dependent processes. J Immunol 1997;159:5197e200. [10] Brincks EL, Katewa A, Kucaba TA, Griffith TS, Legge KL. CD8 T cells utilize TRAIL to control influenza virus infection. J Immunol 2008;181:4918e25. [11] Zhirnov OP, Konakova TE, Wolff T, Klenk HD. NS1 protein of influenza A virus down-regulates apoptosis. J Virol 2002;76:1617e25. [12] Schultz-Cherry S, Dybdahl-Sissoko N, Neumann G, Kawaoka Y, Hinshaw VS. Influenza virus ns1 protein induces apoptosis in cultured cells. J Virol 2001;75:7875e81. [13] Lam WY, Tang JW, Yeung AC, Chiu LC, Sung JJ, et al. Avian influenza virus A/HK/483/97(H5N1) NS1 protein induces apoptosis in human airway epithelial cells. J Virol 2008;82:2741e51. [14] Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, et al. A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 2001;7:1306e12.
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[15] Olsen CW, Kehren JC, Dybdahl-Sissoko NR, Hinshaw VS. bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation. J Virol 1996;70:663e6. [16] Wurzer WJ, Planz O, Ehrhardt C, Giner M, Silberzahn T, et al. Caspase 3 activation is essential for efficient influenza virus propagation. EMBO J 2003;22:2717e28. [17] Yang P, Deng J, Li C, Zhang P, Xing L, et al. Characterization of the 2009 pandemic A/Beijing/501/2009 H1N1 influenza strain in human airway epithelial cells and ferrets. PLoS One 2012;7:e46184. [18] Yang N, Hong X, Yang P, Ju X, Wang Y, et al. The 2009 pandemic A/ Wenshan/01/2009 H1N1 induces apoptotic cell death in human airway epithelial cells. J Mol Cell Biol 2011;3:221e9. [19] Faleiro L, Lazebnik Y. Caspases disrupt the nuclear-cytoplasmic barrier. J Cell Biol 2000;151:951e9. [20] Zhirnov OP, Klenk HD. Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling. Apoptosis 2007;12:1419e32.
Dr. Emma Louise Walton Alex Edelman & Associates 24 rue Ampe`re Malakoff 92240 France E-mail address: [email protected] 23 January 2014 Available online 12 February 2014
