HEPATOLOGY ELSEWHERE
Hepatitis Virus Hijacks Shuttle: Exosome-Like Vesicles Provide Protection Against Neutralizing Antibodies Feng Z, Hensley L, McKnight KL, Hu F, Madden V, Ping L, et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 2013;496:367371. (Reprinted with permission.)
Abstract Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver. Virusspecific antibodies appear only after 3-4 weeks of infection, and typically herald its resolution. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure, when virus replication is well established in the liver. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (‘eHAV’) resemble exosomes, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT), namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between ‘enveloped’ and ‘non-enveloped’ viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.
Comment Viruses come in many forms and shapes that are highly selected by evolution for optimal transmission between hosts and effective infection of host cells.
EDITORS Roberto J. Groszmann, New Haven, CT Yasuko Iwakiri, New Haven, CT Tamar H. Taddei, New Haven, CT
Now emerging evidence suggests that some viruses also can make use of the microvesicle system of host cells for their transmission. Most mammalian cell types are thought to constitutively release small membrane particle microvesicles, including so-called exosomes. Since their discovery 30 years ago,1 these 50 to 100 nm-sized extracellular signaling vesicles are now known to contribute to many (patho)physiological processes, including immunity, coagulation, and bone mineralization.2 Importantly, exosomes can mediate cell-to-cell transmission of genetic information and transfer of messenger RNAs (mRNAs) and microRNAs3 as well as transfer of proteins including interferon-induced antiviral molecules.4 In addition, their role in the transfer of pathogens, pathogen-derived antigens, and virulence factors is emerging.5 Now this recent study by the group of Stanley Lemon from University of North Carolina, Chapel Hill, revealed that the hepatitis A virus (HAV) exploits exosome-like vesicles for its transmission.6 HAV belongs to the group of nonenveloped viruses and is the most common form of acute hepatitis that spreads through fecal-oral transmission. Feng et al.6 identified two distinct populations of viral particles (virions) in HAV-infected hepatoma cell culture supernatant, using a density-gradient: the known capsid virions and a second low-density HAV population that is not detected in a capsid antigen enzyme-linked immunosorbent assay (ELISA). Electron microscopy confirms that this second population of HAV particles is surrounded by membrane structures. The particle size ranges from 50 to 110 nm in diameter, similar to that of exosomes. The authors termed these exosome-like virus particles ‘enveloped HAV’ (eHAV). The exosome-encapsulation of HAV capsids might exert protection of antibody neutralization. To confirm this hypothesis, the authors show that these eHAV are indeed resistant to antibody neutralization and are infectious with an infectivity equivalent to that of nonenveloped virions. The authors showed that in vitro most of the virus released in culture medium is actually eHAV, since the majority of virions contain unprocessed capsid protein VP1pX and mature VP2, whereas the nonenveloped virions contain only fully processed VP1. Also in vivo, in patients with acute hepatitis A infection, the enveloped particles were shown to be the dominant form of HAV detected in serum. 1
2
HEPATOLOGY ELSEWHERE
Confirming the importance of the exosome-pathway in the HAV biogenesis, the authors demonstrated an important role for ESCRT-associated proteins in virus transmission. The ESCRT system is known to be involved in exosome biogenesis in cellular compartments called the multivesicular bodies (MVB).7,8 As a matter of fact, involvement of MVB is considered a hallmark event in exosome biogenesis. Although the involvement of MVB in eHAV production was not formally been proven, the study did show that ESCRT-III binding proteins, VPS4B and ALIX, were involved in the release of eHAV from cells. VPS4B and ALIX were not involved in viral replication or encapsidation of viral RNA. HAV capsid protein VP2 was found to contain YPX1/3L motifs (a motif through which structural proteins interact with ESCRT proteins) which mediate interactions with ALIX. However, it should be noted that the role of ESCRT-III associated proteins may be more complex than just exosome-biogenesis, as knockdown of VPS4b and ALIX also inhibited the release of nonenveloped HAV. Nonenveloped HAV as well as eHAV required entry receptor TIM-1 for viral entry. Interestingly, only eHAV infection was dependent on endosomal acidification, as shown by chloroquine-mediated inhibition, suggesting different postentry steps for the enveloped and nonenveloped HAV. Antibodies directed against the viral capsid effectively neutralize nonenveloped HAV but did not affect eHAV infection. However, immunoglobulin G (IgG) and IgA anti-capsid antibodies did affect eHAV infection when given 6 hours postinoculation. This was not seen with an IgM anticapsid antibody, suggesting eHAV may be neutralized intracellularly after endocytosis of monomeric or dimeric but not pentameric antibodies. The exact mechanism of this observation still needs to be demonstrated, but to some extent it is consistent with the clinical observation that antibody prophylaxis given several days after HAV replication in the liver has been established, still effectively protects against hepatitis A. One of the conclusions of the article is that the classic distinction between enveloped and nonenveloped viruses is blurry, as both category viruses can be cloaked in exosomes or other host derived-membranes. As a wolf in sheep’s clothing, encapsulated viruses can bypass host immune responses. Whether the cloaking of HAV protects viruses from degradation in the gastrointestinal tract and the exterior environment after defecation in contaminated food and drinking water remains to be demonstrated, but is interesting to postulate. HAV is known to be excreted from the liver
HEPATOLOGY, Month 2013
both by way of blood and bile, and exit the host by way of the stool.9 Although the authors showed that exosome-cloaked HAV were able to survive the toxic environment of bile, in feces of infected chimpanzees only nonenveloped virus were identified. Although recent data suggest that RNA packaged in exosomes is protected against degradation in feces,10 there is no direct evidence that the membranes protect the otherwise very stable HAV capsid in the intestinal tract or outside environment. How about a role for exosomes in transmission of other hepatitis viruses? For hepatitis C virus (HCV), our group recently published a study showing that hepatocyte-derived exosomes, isolated from infected hepatoma cells, can contain and transmit virus.11 We showed that exosome-mediated transmission of HCV was partly resistant to antibody neutralization. Earlier studies showed the presence of HCV RNA in bile and fecal samples of HCV-infected individuals,12,13 but the question remained if exosome-encapsulated HCV can act as a protective vehicle for fecal transmission of HCV. For hepatitis E virus (HEV), like HAV a nonenveloped virus, it has been shown that viral particles in the blood of infected persons are associated with membranes.14 Moreover, HEV was shown to utilize ESCRT components for release from infected cells.15 Whether HEV in feces is encapsulated in exosome-like particles, which are protective against degradation in the gastrointestinal tract and beyond, is interesting to postulate but again remains to be demonstrated. VEDASHREE RAMAKRISHNAIAH, M.SC., LUC J.W. VAN DER LAAN, PH.D.
Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
References 1. Harding CV, Heuser JE, Stahl PD. Exosomes: looking back three decades and into the future. J Cell Biol 2013;200:367-371. 2. Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002;2:569-579. 3. Valadi H EK, Bossios A, Sj€ostrand M, Lee JJ, L€otvall JO. Exosomemediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654-659. 4. Li J, Liu K, Liu Y, Xu Y, Zhang F, Yan H, et al. Exosomes mediate the cell-to-cell transmission of IFN-alpha-induced antiviral activity. Nat Immunol 2013;14:793-803. 5. Silverman JM, Reiner NE. Exosomes and other microvesicles in infection biology: organelles with unanticipated phenotypes. Cell Microbiol 2011;13:1-9. 6. Feng Z, Hensley L, McKnight KL, Hu F, Madden V, Ping L, et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 2013;496:367-371.
HEPATOLOGY, Vol. 00, No. 00, 2013
7. Hurley JH, Odorizzi G. Get on the exosome bus with ALIX. 2012;14: 654-655. 8. Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol 2012;14:677-685. 9. Schulman AN, Dienstag JL, Jackson DR, Hoofnagle JH, Gerety RJ, Pucell RH, et al. Hepatitis A antigen particles in liver, bile, and stool of chimpanzees. J Infect Dis 1976;134:80-84. 10. Koga Y, Yasunaga M, Moriya Y, Akasu T, Fujita S, Yamamoto S, et al. Exosome can prevent RNase from degrading microRNA in feces. J Gastrointest Oncol 2011;2:215-222. 11. Ramakrishnaiah V, Thumann C, Fofana I, Habersetzer F, Pan Q, de Ruiter PE, et al. Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells. Proc Natl Acad Sci U S A 2013;110:13109-13113. 12. Haruna Y, Kanda T, Honda M, Takao T, Hayashi N. Detection of hepatitis C virus in the bile and bile duct epithelial cells of hepatitis C virus-infected patients. HEPATOLOGY 2001;33:977-980.
HEPATOLOGY ELSEWHERE
3
13. Beld M, Sentjens R, Rebers S, Weel J, Wertheim van Dillen P, Sol C, et al. Detection and quantitation of hepatitis C virus RNA in feces of chronically infected individuals. J Clin Microbiol 2000;38: 3442-3444. 14. Takahashi M, Tanaka T, Takahashi H, Hosino Y, Nagashima S, Jirintai S, et al. Hepatitis E virus (HEV) strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation. J Clin Microbiol 2010;48:1112-1125. 15. Nagashima S, Takahashi M, Jirintai S, Tanaka T, Nishizawa T, Yasuda J, et al. Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions. J Gen Virol 2011;92:2838-2848. C 2013 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26943 Potential conflict of interest: Nothing to report.
Hepatitis Virus Hijacks Shuttle: Exosome-Like Vesicles Provide Protection Against Neutralizing Antibodies Feng Z, Hensley L, McKnight KL, Hu F, Madden V, Ping L, et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 2013;496:367371. (Reprinted with permission.)
Abstract Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver. Virusspecific antibodies appear only after 3-4 weeks of infection, and typically herald its resolution. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure, when virus replication is well established in the liver. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (‘eHAV’) resemble exosomes, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT), namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between ‘enveloped’ and ‘non-enveloped’ viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.
Comment Viruses come in many forms and shapes that are highly selected by evolution for optimal transmission between hosts and effective infection of host cells.
EDITORS Roberto J. Groszmann, New Haven, CT Yasuko Iwakiri, New Haven, CT Tamar H. Taddei, New Haven, CT
Now emerging evidence suggests that some viruses also can make use of the microvesicle system of host cells for their transmission. Most mammalian cell types are thought to constitutively release small membrane particle microvesicles, including so-called exosomes. Since their discovery 30 years ago,1 these 50 to 100 nm-sized extracellular signaling vesicles are now known to contribute to many (patho)physiological processes, including immunity, coagulation, and bone mineralization.2 Importantly, exosomes can mediate cell-to-cell transmission of genetic information and transfer of messenger RNAs (mRNAs) and microRNAs3 as well as transfer of proteins including interferon-induced antiviral molecules.4 In addition, their role in the transfer of pathogens, pathogen-derived antigens, and virulence factors is emerging.5 Now this recent study by the group of Stanley Lemon from University of North Carolina, Chapel Hill, revealed that the hepatitis A virus (HAV) exploits exosome-like vesicles for its transmission.6 HAV belongs to the group of nonenveloped viruses and is the most common form of acute hepatitis that spreads through fecal-oral transmission. Feng et al.6 identified two distinct populations of viral particles (virions) in HAV-infected hepatoma cell culture supernatant, using a density-gradient: the known capsid virions and a second low-density HAV population that is not detected in a capsid antigen enzyme-linked immunosorbent assay (ELISA). Electron microscopy confirms that this second population of HAV particles is surrounded by membrane structures. The particle size ranges from 50 to 110 nm in diameter, similar to that of exosomes. The authors termed these exosome-like virus particles ‘enveloped HAV’ (eHAV). The exosome-encapsulation of HAV capsids might exert protection of antibody neutralization. To confirm this hypothesis, the authors show that these eHAV are indeed resistant to antibody neutralization and are infectious with an infectivity equivalent to that of nonenveloped virions. The authors showed that in vitro most of the virus released in culture medium is actually eHAV, since the majority of virions contain unprocessed capsid protein VP1pX and mature VP2, whereas the nonenveloped virions contain only fully processed VP1. Also in vivo, in patients with acute hepatitis A infection, the enveloped particles were shown to be the dominant form of HAV detected in serum. 1
2
HEPATOLOGY ELSEWHERE
Confirming the importance of the exosome-pathway in the HAV biogenesis, the authors demonstrated an important role for ESCRT-associated proteins in virus transmission. The ESCRT system is known to be involved in exosome biogenesis in cellular compartments called the multivesicular bodies (MVB).7,8 As a matter of fact, involvement of MVB is considered a hallmark event in exosome biogenesis. Although the involvement of MVB in eHAV production was not formally been proven, the study did show that ESCRT-III binding proteins, VPS4B and ALIX, were involved in the release of eHAV from cells. VPS4B and ALIX were not involved in viral replication or encapsidation of viral RNA. HAV capsid protein VP2 was found to contain YPX1/3L motifs (a motif through which structural proteins interact with ESCRT proteins) which mediate interactions with ALIX. However, it should be noted that the role of ESCRT-III associated proteins may be more complex than just exosome-biogenesis, as knockdown of VPS4b and ALIX also inhibited the release of nonenveloped HAV. Nonenveloped HAV as well as eHAV required entry receptor TIM-1 for viral entry. Interestingly, only eHAV infection was dependent on endosomal acidification, as shown by chloroquine-mediated inhibition, suggesting different postentry steps for the enveloped and nonenveloped HAV. Antibodies directed against the viral capsid effectively neutralize nonenveloped HAV but did not affect eHAV infection. However, immunoglobulin G (IgG) and IgA anti-capsid antibodies did affect eHAV infection when given 6 hours postinoculation. This was not seen with an IgM anticapsid antibody, suggesting eHAV may be neutralized intracellularly after endocytosis of monomeric or dimeric but not pentameric antibodies. The exact mechanism of this observation still needs to be demonstrated, but to some extent it is consistent with the clinical observation that antibody prophylaxis given several days after HAV replication in the liver has been established, still effectively protects against hepatitis A. One of the conclusions of the article is that the classic distinction between enveloped and nonenveloped viruses is blurry, as both category viruses can be cloaked in exosomes or other host derived-membranes. As a wolf in sheep’s clothing, encapsulated viruses can bypass host immune responses. Whether the cloaking of HAV protects viruses from degradation in the gastrointestinal tract and the exterior environment after defecation in contaminated food and drinking water remains to be demonstrated, but is interesting to postulate. HAV is known to be excreted from the liver
HEPATOLOGY, Month 2013
both by way of blood and bile, and exit the host by way of the stool.9 Although the authors showed that exosome-cloaked HAV were able to survive the toxic environment of bile, in feces of infected chimpanzees only nonenveloped virus were identified. Although recent data suggest that RNA packaged in exosomes is protected against degradation in feces,10 there is no direct evidence that the membranes protect the otherwise very stable HAV capsid in the intestinal tract or outside environment. How about a role for exosomes in transmission of other hepatitis viruses? For hepatitis C virus (HCV), our group recently published a study showing that hepatocyte-derived exosomes, isolated from infected hepatoma cells, can contain and transmit virus.11 We showed that exosome-mediated transmission of HCV was partly resistant to antibody neutralization. Earlier studies showed the presence of HCV RNA in bile and fecal samples of HCV-infected individuals,12,13 but the question remained if exosome-encapsulated HCV can act as a protective vehicle for fecal transmission of HCV. For hepatitis E virus (HEV), like HAV a nonenveloped virus, it has been shown that viral particles in the blood of infected persons are associated with membranes.14 Moreover, HEV was shown to utilize ESCRT components for release from infected cells.15 Whether HEV in feces is encapsulated in exosome-like particles, which are protective against degradation in the gastrointestinal tract and beyond, is interesting to postulate but again remains to be demonstrated. VEDASHREE RAMAKRISHNAIAH, M.SC., LUC J.W. VAN DER LAAN, PH.D.
Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
References 1. Harding CV, Heuser JE, Stahl PD. Exosomes: looking back three decades and into the future. J Cell Biol 2013;200:367-371. 2. Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002;2:569-579. 3. Valadi H EK, Bossios A, Sj€ostrand M, Lee JJ, L€otvall JO. Exosomemediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654-659. 4. Li J, Liu K, Liu Y, Xu Y, Zhang F, Yan H, et al. Exosomes mediate the cell-to-cell transmission of IFN-alpha-induced antiviral activity. Nat Immunol 2013;14:793-803. 5. Silverman JM, Reiner NE. Exosomes and other microvesicles in infection biology: organelles with unanticipated phenotypes. Cell Microbiol 2011;13:1-9. 6. Feng Z, Hensley L, McKnight KL, Hu F, Madden V, Ping L, et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 2013;496:367-371.
HEPATOLOGY, Vol. 00, No. 00, 2013
7. Hurley JH, Odorizzi G. Get on the exosome bus with ALIX. 2012;14: 654-655. 8. Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol 2012;14:677-685. 9. Schulman AN, Dienstag JL, Jackson DR, Hoofnagle JH, Gerety RJ, Pucell RH, et al. Hepatitis A antigen particles in liver, bile, and stool of chimpanzees. J Infect Dis 1976;134:80-84. 10. Koga Y, Yasunaga M, Moriya Y, Akasu T, Fujita S, Yamamoto S, et al. Exosome can prevent RNase from degrading microRNA in feces. J Gastrointest Oncol 2011;2:215-222. 11. Ramakrishnaiah V, Thumann C, Fofana I, Habersetzer F, Pan Q, de Ruiter PE, et al. Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells. Proc Natl Acad Sci U S A 2013;110:13109-13113. 12. Haruna Y, Kanda T, Honda M, Takao T, Hayashi N. Detection of hepatitis C virus in the bile and bile duct epithelial cells of hepatitis C virus-infected patients. HEPATOLOGY 2001;33:977-980.
HEPATOLOGY ELSEWHERE
3
13. Beld M, Sentjens R, Rebers S, Weel J, Wertheim van Dillen P, Sol C, et al. Detection and quantitation of hepatitis C virus RNA in feces of chronically infected individuals. J Clin Microbiol 2000;38: 3442-3444. 14. Takahashi M, Tanaka T, Takahashi H, Hosino Y, Nagashima S, Jirintai S, et al. Hepatitis E virus (HEV) strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation. J Clin Microbiol 2010;48:1112-1125. 15. Nagashima S, Takahashi M, Jirintai S, Tanaka T, Nishizawa T, Yasuda J, et al. Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions. J Gen Virol 2011;92:2838-2848. C 2013 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26943 Potential conflict of interest: Nothing to report.
