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Autophagy, Apoptosis, and the Influenza Virus M2 Protein
Cell Host & Microbe
Previews Autophagy, Apoptosis, and the Influenza Virus M2 Protein Jeremy S. Rossman1,2 and Robert A. Lamb1,2,* 1Department
of Biochemistry, Molecular Biology, and Cell Biology Hughes Medical Institute Northwestern University, Evanston, IL 60208-3500, USA *Correspondence: [email protected] DOI 10.1016/j.chom.2009.09.009 2Howard
Viral subversion and inhibition of host cell autophagy has been documented for several viruses. In this issue of Cell Host & Microbe, Gannage´ et al. (2009) show that the influenza virus M2 integral membrane protein blocks autophagosome maturation, significantly affecting host cell apoptosis. Autophagy is one of the main pathways for protein degradation within the cell. Autophagy begins with the activation of phosphatidylinositol 3-kinase (PI3K) and the autophagy-related protein (Atg) 6 (also named Beclin-1) by a wide variety of targets and signaling pathways. Two Atg protein complexes then modify Atg8 (also named LC3), mediating the assembly of a double-membrane isolation crescent around the target protein. The crescent-shaped membrane then seals into a spherical vesicle, enabling fusion with the lysosome, resulting in content degradation by lysosomal proteases (reviewed in Deretic and Levine, 2009) (Figure 1). While the autophagy pathway serves the main function of degrading large protein complexes, additional functions have been found that suggest a role for autophagy in the innate and adaptive immune response. Autophagy of proteins from infecting organisms leads to protein degradation, with the resulting peptide fragments becoming complexed with MHC for presentation to the adaptive immune system or presentation to intracellular receptors of the innate immune system following autophagosome fusion with the endosome. Autophagy also acts directly as a measure of innate immunity by engulfing and degrading entire infecting organisms, such as herpes simplex virus 1 (HSV-1). To combat this process, many pathogens encode proteins that block the induction of autophagy or the maturation of autophagosomes. In the case of herpesviruses, HSV-1, Kaposi’s sarcoma-associated herpesvirus (KSHV), and mouse herpesvirus 68 (MHV-68) encode proteins that bind to Beclin-1, preventing the initiation of autophagy (reviewed in Deretic
and Levine, 2009). Additionally, HIV-1 was recently found to encode a viral protein capable of blocking autophagosome-lysosome fusion, preventing virion degradation by autophagy (Kyei et al., 2009). Several RNA viruses have been shown to induce autophagy and to block the maturation of autophagosomes, possibly utilizing the resulting immature autophagosome for their own replication (reviewed in Deretic and Levine, 2009). Replication in the enclosed space of the autophagosome may serve to concentrate viral proteins, increasing the efficiency of replication. Alternatively, replication in the autophagosome may shield the viral genome and associated proteins from detection by the innate immune system. Additionally, as has been shown for poliovirus, virions within the autophagosome can be directly shuttled out of the cell as the autophagosome fuses with the plasma membrane, allowing for the nonlytic release of assembled virions (reviewed in Deretic and Levine, 2009). The article by Gannage´ et al. in this issue of Cell Host & Microbe adds influenza virus to the growing list of viruses that block autophagosome maturation (Gannage´ et al., 2009). However, in contrast to other RNA viruses that may utilize the autophagosome as a site of replication, influenza virus appears to retain its nuclear-cytoplasmic replication, despite activating and modulating autophagy. In searching for the viral mediator of the autophagy block, the authors find a surprising role for the influenza virus M2 protein. M2 functions as a proton-selective ion channel and is responsible for acidification of the viral core upon receptor-mediated endocytosis, allowing
for release of the viral genome and associated proteins into the cytoplasm (reviewed in Pinto and Lamb, 2006). Additionally, M2 was shown to have a crucial role in influenza virus assembly and budding (Chen et al., 2008). Gannage´ et al. now find that expression of the M2 protein blocks the normal maturation of autophagosomes, preventing their fusion with the lysosome and resulting in the generation of a single large perinuclear autophagosome. This block of autophagosome maturation does not involve M2 ion channel activity, as it occurred in the presence of the channel inhibitor, amantadine. Gannage´ et al. show that the first 60 amino acids of M2 enable binding to Beclin-1 and are sufficient for inhibition of autophagy, though the specific mechanism of inhibition was not determined in this study. Given that M2 residues 25–42 function as the membrane-spanning pore of the ion channel, Beclin-1 binding and autophagy modulation may be mapped to either the luminally exposed, highly conserved ectodomain (residues 1–24) or to the cytoplasmic amphipathic helix (residues 46–62). It is not clear if M2 binding to Beclin-1 disrupts a crucial Beclin-1 complex or if the modulation of autophagy requires a more active role of the M2 protein. Beclin-1 has recently been shown to interact with two different protein complexes regulating both the initiation of autophagy as well as autophagosome-lysosome fusion (Matsunaga et al., 2009), and multiple viral proteins have been found that bind to Beclin-1, blocking autophagy initiation (HSV-1) (reviewed in Deretic and Levine, 2009) as well as autophagosome maturation (HIV-1) (Kyei et al., 2009; reviewed in Deretic and Levine, 2009). The
Cell Host & Microbe 6, October 22, 2009 ª2009 Elsevier Inc. 299
Cell Host & Microbe
Previews exact mechanism of autophreplication when they inhibited agy induction was not deterautophagy in the MEF cell line. mined in this influenza virus There appears to be a delistudy, but it is possible that M2 cate balance between the influenza virus-mediated inducmay trigger the initiation of tion of autophagy and the autophagy as well as inhibiting autophagosome maturation, induction of apoptosis, with perhaps all mediated through a high degree of crosstalk the interaction with Beclin-1. between the two pathways. For many of the viruses that This balance may be skewed block autophagosome matuwhen examining different cell ration, inhibition of autophagy types, and its ultimate effect significantly affects viral replion viral replication may be cation (Kyei et al., 2009; reclarified only when examined viewed in Deretic and Levine, in the context of the host 2009). However, Gannage´ immune system during an et al. observed that, for influin vivo infection. The work by Figure 1. The Influenza Virus M2 Protein Blocks Autophagosomeenza virus, replication proGannage´ et al. further supLysosome Fusion, Inhibiting the Completion of Autophagy and gressed normally even in an ports the notion that M2 is a Triggering the Induction of Apoptosis autophagy-deficient cell line, multifunctional protein whose suggesting that viral replication does not possible that influenza virus is able to modification of host autophagy and aporequire the autophagosome nor does specifically modify the extent and pro- ptosis may have considerable affects on autophagy prevent viral replication. Intrigu- gression of apoptosis, and the M2 protein the in vivo replication and spread of influingly, though, the authors find a strong may be a key player in this process (Gan- enza virus. relationship between autophagy inhibition nage´ et al., 2009). and the induction of apoptosis. Influenza There are many interesting questions ACKNOWLEDGMENTS virus infections have been shown to stemming from the research of Gannage´ induce apoptosis, a process that is closely et al., and their resolution may help to Work in our laboratory is supported by research grant R01 AI-20201 (R.A.L.) from the National Institutes of regulated by multiple viral proteins, both further elucidate the role of influenza virus Allergy and Infectious Diseases. R.A.L. is an Investipro- and antiapoptotic (reviewed in Lud- M2 in autophagy and the role of autophagy gator of the Howard Hughes Medical Institute. wig et al., 2006). Blockade of premature and apoptosis in viral infection. It was apoptosis appears to be essential for viral shown that complete inhibition of autoph- REFERENCES replication, while the induction of apo- agy (by Atg5 deletion) increases influenza Boya, P., Gonza´lez-Polo, R.A., Casares, N., Perfetptosis may be involved in evasion of the virus-induced apoptosis, whereas permit- tini, J.L., Dessen, P., Larochette, N., Me´tivier, D., immune system. Gannage´ et al. show ting autophagy to progress (by M2 deletion) Meley, D., Souquere, S., Yoshimori, T., et al. that complete inhibition of the autophagy decreases influenza virus-induced apo- (2005). Mol. Cell. Biol. 25, 1025–1040. pathway significantly increases apoptosis ptosis. It is not clear why influenza virus Chen, B.J., Leser, G.P., Jackson, D., and Lamb, in influenza virus-infected cells (Figure 1). would strive to induce this moderate level R.A. (2008). J. Virol. 82, 10059–10070. This increase in apoptosis was not corre- of apoptosis and what the effect would be Deretic, V., and Levine, B. (2009). Cell Host lated with any change in viral titer, sug- of shifting the balance to one extreme or Microbe 5, 527–549. gesting that the tradeoff between autoph- the other. Additionally, there appears to Gannage´, M., Schmid, D., Albrecht, R., Dengjel, J., agy and apoptosis does not affect viral be cell-type dependence for the extent of Torossi, T., Ra¨mer, P.C., Lee, M., Strowig, T., replication in a tissue culture model. How- autophagy and apoptosis induction that Arrey, F., Conenello, G., et al. (2009). Cell Host Microbe 6, this issue, 367–380. ever, the effects of increased apoptosis in may allow for selective depletion of iman animal model of influenza virus infec- mune cells while inducing the prolonged Kyei, G.B., Dinkins, C., Davis, A.S., Roberts, E., Singh, S.B., Dong, C., Wu, L., Kominami, E., Ueno, T., Yamation, where the host immune system is survival of other cell types, enhancing viral moto, A., et al. (2009). J. Cell Biol. 186, 255–268. active, could be significant. In this study, replication. Recent work has shown that Gannage´ et al. saw a significant increase inhibition of autophagy in A549 cells (as Ludwig, S., Pleschka, S., Planz, O., and Wolff, T. (2006). Cell. Microbiol. 8, 375–386. in the amount of viral protein and viral opposed to the MEF cell line employed by RNA released from autophagy-defective Gannage´ et al.) reduces the replication of Matsunaga, K., Saitoh, T., Tabata, K., Omori, H., Satoh, T., Kurotori, N., Maejima, I., Shirahamacells. This released protein could serve influenza virus (Zhou et al., 2009). Addi- Noda, K., Ichimura, T., Isobe, T., et al. (2009). to activate the host immune response, tional work in the MEF cell line has shown Nat. Cell Biol. 11, 385–396. limiting viral replication and spread. Along that influenza virus does not induce au- McLean, J.E., Datan, E., Matassov, D., and Zakeri, these lines, inhibition of autophagy pro- tophagy unless apoptosis is first inhibited Z.F. (2009). J. Virol. 83, 8233–8246. gression, specifically block of autophago- (McLean et al., 2009), thus supporting a Pinto, L.H., and Lamb, R.A. (2006). J. Biol. Chem. some-lysosome fusion, has been shown physiologic link between the two pro- 281, 8997–9000. to induce apoptosis (Boya et al., 2005). In cesses during the viral life cycle and proZhou, Z., Jiang, X., Liu, D., Fan, Z., Hu, X., Yan, J., considering that deletion of the M2 protein viding a possible explanation for why Wang, M., and Gao, G.F. (2009). Autophagy 5, decreased infected cell apoptosis, it is Gannage´ et al. did not see any effect on viral 321–328. 300 Cell Host & Microbe 6, October 22, 2009 ª2009 Elsevier Inc.
Previews Autophagy, Apoptosis, and the Influenza Virus M2 Protein Jeremy S. Rossman1,2 and Robert A. Lamb1,2,* 1Department
of Biochemistry, Molecular Biology, and Cell Biology Hughes Medical Institute Northwestern University, Evanston, IL 60208-3500, USA *Correspondence: [email protected] DOI 10.1016/j.chom.2009.09.009 2Howard
Viral subversion and inhibition of host cell autophagy has been documented for several viruses. In this issue of Cell Host & Microbe, Gannage´ et al. (2009) show that the influenza virus M2 integral membrane protein blocks autophagosome maturation, significantly affecting host cell apoptosis. Autophagy is one of the main pathways for protein degradation within the cell. Autophagy begins with the activation of phosphatidylinositol 3-kinase (PI3K) and the autophagy-related protein (Atg) 6 (also named Beclin-1) by a wide variety of targets and signaling pathways. Two Atg protein complexes then modify Atg8 (also named LC3), mediating the assembly of a double-membrane isolation crescent around the target protein. The crescent-shaped membrane then seals into a spherical vesicle, enabling fusion with the lysosome, resulting in content degradation by lysosomal proteases (reviewed in Deretic and Levine, 2009) (Figure 1). While the autophagy pathway serves the main function of degrading large protein complexes, additional functions have been found that suggest a role for autophagy in the innate and adaptive immune response. Autophagy of proteins from infecting organisms leads to protein degradation, with the resulting peptide fragments becoming complexed with MHC for presentation to the adaptive immune system or presentation to intracellular receptors of the innate immune system following autophagosome fusion with the endosome. Autophagy also acts directly as a measure of innate immunity by engulfing and degrading entire infecting organisms, such as herpes simplex virus 1 (HSV-1). To combat this process, many pathogens encode proteins that block the induction of autophagy or the maturation of autophagosomes. In the case of herpesviruses, HSV-1, Kaposi’s sarcoma-associated herpesvirus (KSHV), and mouse herpesvirus 68 (MHV-68) encode proteins that bind to Beclin-1, preventing the initiation of autophagy (reviewed in Deretic
and Levine, 2009). Additionally, HIV-1 was recently found to encode a viral protein capable of blocking autophagosome-lysosome fusion, preventing virion degradation by autophagy (Kyei et al., 2009). Several RNA viruses have been shown to induce autophagy and to block the maturation of autophagosomes, possibly utilizing the resulting immature autophagosome for their own replication (reviewed in Deretic and Levine, 2009). Replication in the enclosed space of the autophagosome may serve to concentrate viral proteins, increasing the efficiency of replication. Alternatively, replication in the autophagosome may shield the viral genome and associated proteins from detection by the innate immune system. Additionally, as has been shown for poliovirus, virions within the autophagosome can be directly shuttled out of the cell as the autophagosome fuses with the plasma membrane, allowing for the nonlytic release of assembled virions (reviewed in Deretic and Levine, 2009). The article by Gannage´ et al. in this issue of Cell Host & Microbe adds influenza virus to the growing list of viruses that block autophagosome maturation (Gannage´ et al., 2009). However, in contrast to other RNA viruses that may utilize the autophagosome as a site of replication, influenza virus appears to retain its nuclear-cytoplasmic replication, despite activating and modulating autophagy. In searching for the viral mediator of the autophagy block, the authors find a surprising role for the influenza virus M2 protein. M2 functions as a proton-selective ion channel and is responsible for acidification of the viral core upon receptor-mediated endocytosis, allowing
for release of the viral genome and associated proteins into the cytoplasm (reviewed in Pinto and Lamb, 2006). Additionally, M2 was shown to have a crucial role in influenza virus assembly and budding (Chen et al., 2008). Gannage´ et al. now find that expression of the M2 protein blocks the normal maturation of autophagosomes, preventing their fusion with the lysosome and resulting in the generation of a single large perinuclear autophagosome. This block of autophagosome maturation does not involve M2 ion channel activity, as it occurred in the presence of the channel inhibitor, amantadine. Gannage´ et al. show that the first 60 amino acids of M2 enable binding to Beclin-1 and are sufficient for inhibition of autophagy, though the specific mechanism of inhibition was not determined in this study. Given that M2 residues 25–42 function as the membrane-spanning pore of the ion channel, Beclin-1 binding and autophagy modulation may be mapped to either the luminally exposed, highly conserved ectodomain (residues 1–24) or to the cytoplasmic amphipathic helix (residues 46–62). It is not clear if M2 binding to Beclin-1 disrupts a crucial Beclin-1 complex or if the modulation of autophagy requires a more active role of the M2 protein. Beclin-1 has recently been shown to interact with two different protein complexes regulating both the initiation of autophagy as well as autophagosome-lysosome fusion (Matsunaga et al., 2009), and multiple viral proteins have been found that bind to Beclin-1, blocking autophagy initiation (HSV-1) (reviewed in Deretic and Levine, 2009) as well as autophagosome maturation (HIV-1) (Kyei et al., 2009; reviewed in Deretic and Levine, 2009). The
Cell Host & Microbe 6, October 22, 2009 ª2009 Elsevier Inc. 299
Cell Host & Microbe
Previews exact mechanism of autophreplication when they inhibited agy induction was not deterautophagy in the MEF cell line. mined in this influenza virus There appears to be a delistudy, but it is possible that M2 cate balance between the influenza virus-mediated inducmay trigger the initiation of tion of autophagy and the autophagy as well as inhibiting autophagosome maturation, induction of apoptosis, with perhaps all mediated through a high degree of crosstalk the interaction with Beclin-1. between the two pathways. For many of the viruses that This balance may be skewed block autophagosome matuwhen examining different cell ration, inhibition of autophagy types, and its ultimate effect significantly affects viral replion viral replication may be cation (Kyei et al., 2009; reclarified only when examined viewed in Deretic and Levine, in the context of the host 2009). However, Gannage´ immune system during an et al. observed that, for influin vivo infection. The work by Figure 1. The Influenza Virus M2 Protein Blocks Autophagosomeenza virus, replication proGannage´ et al. further supLysosome Fusion, Inhibiting the Completion of Autophagy and gressed normally even in an ports the notion that M2 is a Triggering the Induction of Apoptosis autophagy-deficient cell line, multifunctional protein whose suggesting that viral replication does not possible that influenza virus is able to modification of host autophagy and aporequire the autophagosome nor does specifically modify the extent and pro- ptosis may have considerable affects on autophagy prevent viral replication. Intrigu- gression of apoptosis, and the M2 protein the in vivo replication and spread of influingly, though, the authors find a strong may be a key player in this process (Gan- enza virus. relationship between autophagy inhibition nage´ et al., 2009). and the induction of apoptosis. Influenza There are many interesting questions ACKNOWLEDGMENTS virus infections have been shown to stemming from the research of Gannage´ induce apoptosis, a process that is closely et al., and their resolution may help to Work in our laboratory is supported by research grant R01 AI-20201 (R.A.L.) from the National Institutes of regulated by multiple viral proteins, both further elucidate the role of influenza virus Allergy and Infectious Diseases. R.A.L. is an Investipro- and antiapoptotic (reviewed in Lud- M2 in autophagy and the role of autophagy gator of the Howard Hughes Medical Institute. wig et al., 2006). Blockade of premature and apoptosis in viral infection. It was apoptosis appears to be essential for viral shown that complete inhibition of autoph- REFERENCES replication, while the induction of apo- agy (by Atg5 deletion) increases influenza Boya, P., Gonza´lez-Polo, R.A., Casares, N., Perfetptosis may be involved in evasion of the virus-induced apoptosis, whereas permit- tini, J.L., Dessen, P., Larochette, N., Me´tivier, D., immune system. Gannage´ et al. show ting autophagy to progress (by M2 deletion) Meley, D., Souquere, S., Yoshimori, T., et al. that complete inhibition of the autophagy decreases influenza virus-induced apo- (2005). Mol. Cell. Biol. 25, 1025–1040. pathway significantly increases apoptosis ptosis. It is not clear why influenza virus Chen, B.J., Leser, G.P., Jackson, D., and Lamb, in influenza virus-infected cells (Figure 1). would strive to induce this moderate level R.A. (2008). J. Virol. 82, 10059–10070. This increase in apoptosis was not corre- of apoptosis and what the effect would be Deretic, V., and Levine, B. (2009). Cell Host lated with any change in viral titer, sug- of shifting the balance to one extreme or Microbe 5, 527–549. gesting that the tradeoff between autoph- the other. Additionally, there appears to Gannage´, M., Schmid, D., Albrecht, R., Dengjel, J., agy and apoptosis does not affect viral be cell-type dependence for the extent of Torossi, T., Ra¨mer, P.C., Lee, M., Strowig, T., replication in a tissue culture model. How- autophagy and apoptosis induction that Arrey, F., Conenello, G., et al. (2009). Cell Host Microbe 6, this issue, 367–380. ever, the effects of increased apoptosis in may allow for selective depletion of iman animal model of influenza virus infec- mune cells while inducing the prolonged Kyei, G.B., Dinkins, C., Davis, A.S., Roberts, E., Singh, S.B., Dong, C., Wu, L., Kominami, E., Ueno, T., Yamation, where the host immune system is survival of other cell types, enhancing viral moto, A., et al. (2009). J. Cell Biol. 186, 255–268. active, could be significant. In this study, replication. Recent work has shown that Gannage´ et al. saw a significant increase inhibition of autophagy in A549 cells (as Ludwig, S., Pleschka, S., Planz, O., and Wolff, T. (2006). Cell. Microbiol. 8, 375–386. in the amount of viral protein and viral opposed to the MEF cell line employed by RNA released from autophagy-defective Gannage´ et al.) reduces the replication of Matsunaga, K., Saitoh, T., Tabata, K., Omori, H., Satoh, T., Kurotori, N., Maejima, I., Shirahamacells. This released protein could serve influenza virus (Zhou et al., 2009). Addi- Noda, K., Ichimura, T., Isobe, T., et al. (2009). to activate the host immune response, tional work in the MEF cell line has shown Nat. Cell Biol. 11, 385–396. limiting viral replication and spread. Along that influenza virus does not induce au- McLean, J.E., Datan, E., Matassov, D., and Zakeri, these lines, inhibition of autophagy pro- tophagy unless apoptosis is first inhibited Z.F. (2009). J. Virol. 83, 8233–8246. gression, specifically block of autophago- (McLean et al., 2009), thus supporting a Pinto, L.H., and Lamb, R.A. (2006). J. Biol. Chem. some-lysosome fusion, has been shown physiologic link between the two pro- 281, 8997–9000. to induce apoptosis (Boya et al., 2005). In cesses during the viral life cycle and proZhou, Z., Jiang, X., Liu, D., Fan, Z., Hu, X., Yan, J., considering that deletion of the M2 protein viding a possible explanation for why Wang, M., and Gao, G.F. (2009). Autophagy 5, decreased infected cell apoptosis, it is Gannage´ et al. did not see any effect on viral 321–328. 300 Cell Host & Microbe 6, October 22, 2009 ª2009 Elsevier Inc.