VIROLOGY
Delineating Ebola entry By Darryl Falzarano1 and Heinz Feldmann2
ILLUSTRATION: ADAPTED BY C. BICKEL/SCIENCE
T
tinic acid adenine dinucleotide phosphate (NAADP)–stimulated intracellular calcium channels—known as the TPCs. These channels are mainly localized to endosomes and lysosomes (acidic compartments where contents are degraded). Through the use of cells lacking TPC2, small interfering RNA, and small-molecule inhibitors, Sakurai et al. determined that TPC2 is required for Ebola entry. Moreover, the requirement for
he means by which Ebola virus enters a cell are becoming less mysterious. Although a definitive cell surface receptor for the virus, if there is one, remains to be identified, the mechanism of gaining entry is beginning to be fleshed out. Once inside the cell, the importance of numerous sequential processes is becoming better understood. On page 995 of this issue, Sakurai et al. (1) add another element to the viral entry pathway by showing that a calcium channel called two-pore channel 2 (TPC2) is required for Ebola virus release of the viral genome into the host cell. After Ebola’s surface glycoprotein binds to receptors, which may be nonspecific and possibly comprise numerous carbohydrate proteins (2), the virus enters the cell via macropinocytosis, a nonselective process of engulfment (3, 4) (see the figure). Once internalized into Endosome a membrane-bound vesicle (endosome), Ebola glycoproteins are cleaved (5) while being exposed to an increasingly acidic and reducing environment. All this occurs in the endosome where an essential interaction with a protein called Niemann-Pick C1 (NPC1) was proposed to result in the release of the virus’s Late endosome genetic material in a process known as fusion (6, 7). A small interfering RNA screen identified calcium signaling in the host cell, among other events, as necessary for Ebola virus entry (8). L-type calcium channels were NPC1 initially pinpointed as the key element involved. However, one of Lysosome? five compounds that block these channels did not prevent Ebola infection, suggesting that another mechanism is also involved. The effective inhibitors also block nico-
TPC2 was shown to be specific to the glycoprotein of Ebola virus, suggesting a highly specific endosomal processing pathway. Previous work demonstrated that NPC1 is also essential for Ebola virus entry and proposed that it may function as an endosomal receptor that triggers fusion (7). However, the findings of Sakurai et al. suggest that NPC1 is not likely required for fusion itself because late endosomes expressing TPC2, but not NPC1, correlated with productive infection in cultured cells. This indicates that interaction Receptor of the cleaved glycoprotein with (carbohydrate-binding protein) NPC1 occurs before the glycoprotein interaction with TPC2 and prior to fusion. Sakurai et al. further demonstrate the relevance of TPC2 by Macropinocytosis blocking the channel’s activity with the drug tetrandrine, which improved survival of Ebola-infected mice. This supports the important role of TPC2 for Ebola virus infection; however, it does not indicate that a viable treatment is close at hand. The partial Cellular effectiveness of the drug in mice protease was further reduced by delaying treatment by 1 day. This questions whether any protection would be observed in a macaque model (the “gold standard” for Ebola drug Tetrandrine efficacy testing) (9). Tetrandrine, (blocks infection) originally a traditional Chinese medicine (found in the plant Stephania tetrandra), is not apTPC2 proved for use in humans (except NAADP in China). In addition, the dose given to mice by Sakurai et al. was many times the half-maximal inhibitory dose observed in tissue 2+ Ca culture, and likely exceeds doses that would be considered safe for humans. Given the mode of action, it seems unlikely that tetranTPC2 drine treatment would be superior to the most advanced Ebola postCa2+
1
Vaccine and Infectious Disease Organization– International Vaccine Centre, University of Saskatchewan, Saskatoon, SK Canada. 2Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Hamilton, MT, USA. E-mail: [email protected]
Fusion Genome release Virus replication
SCIENCE sciencemag.org
Ebola’s entry. A model of infection shows that once the virus is internalized into an endosome, the viral glycoprotein is cleaved and binds to NPC1. The calcium channel TPC2 is then activated prior to a fusion event that releases the viral genome into the cell. The drug tetrandrine blocks TPC2. 27 FEBRUARY 2015 • VOL 347 ISSUE 6225
Published by AAAS
947
Downloaded from www.sciencemag.org on February 26, 2015
A specific calcium channel is required for Ebola virus to infect a cell
INSIGHTS | P E R S P E C T I V E S
REFERENCES
1. Y. Sakurai et al., Science 347, 995 (2015). 2. C. L. Hunt, N. J. Lennemann, W. Maury, Viruses 4, 258 (2012). 3. M. F. Saeed, A. A. Kolokoltsov, T. Albrecht, R. A. Davey, PLOS Pathog. 6, e1001110 (2010). 4. A. Nanbo et al., PLOS Pathog. 6, e1001121 (2010). 5. K. Chandran, N. J. Sullivan, U. Felbor, S. P. Whelan, J. M. Cunningham, Science 308, 1643 (2005). 6. J. E. Carette et al., Nature 477, 340 (2011). 7. M. Côté et al., Nature 477, 344 (2011). 8. A. A. Kolokoltsov, M. F. Saeed, A. N. Freiberg, M. R. Holbrook, R. A. Davey, Drug Dev. Res. 70, 255 (2009). 9. X. Qiu et al., Nature 514, 47 (2014). 10. E. Picazo, F. Giordanetto, Drug Discov. Today 10.1016/j. drudis.2014.12.010 (2014). ACKNOWLEDGMENTS
This work was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. 10.1126/science.aaa8121
948
Female avoids male harassment Male produces steroid hormone 20E and forms a mating plug
Female receives 20E, which interacts with the MISO protein to stimulate egg production and decrease remating
Male fathers more ofspring
Male
Reduced immune response allows plasmodium infection Female
Coevolved sexual traits in male and female mosquitoes. Mitchell et al. show that as anophelene mosquitoes evolved, so have the traits that affect interactions between the sexes, and likely also their capacity as vectors of human malaria.
EVOLUTION
An unexpected cost of sex Coevolution of male and female mosquitoes influences whether mosquitoes transmit human malaria By Suzanne H. Alonzo
S
election arising from interactions between the sexes is responsible for some of the most striking diversity on the planet. These interactions generate coevolutionary dynamics between males and females that have shaped traits such as the striking courtship displays of male birds and the less winsome mating appendages of some male insects (1). But research on sexual selection has relevance beyond understanding the weird sex lives of animals. For example, human disturbance of sexual selection can lead to the loss of native species (2) and sexually selected male harassment of females can increase a species’ risk of extinction (3). On page 985 of this issue, Mitchell et al. (4) show that sexual selection can also be relevant to human health. The authors report evidence of a coevolved suite of male and female sexual traits in the mosquitoes that transmit the vector responsible for human malaria (see the figure). These traits include the evolution of a “mating plug” that allows males to transfer not only sperm but also the steroid hormone 20-hydroxyecdysone (20E) to females. In the female’s reproductive tract, 20E sets off a cascade of processes that increase egg production and decrease the rate
at which females mate with other males. Yet, this only happens in species where these male and female traits have coevolved. Mitchell et al. also demonstrate rapid evolution of the mating-induced stimulator of oogenesis (MISO) protein, which is produced by females and interacts with 20E inside the female to increase egg production. In the main vector of malaria, Anopheles gambiae, proteins that stimulate egg production are known to decrease female innate immunity, which increases the susceptibility of female mosquitoes to infection by the plasmodium that causes malaria (5). It is not currently known why this suite of traits arose in some but not all mosquitoes and if their presence can be used to predict whether a species will transmit human malaria. Mosquitoes are not exceptional in having a suite of coevolved traits that suggest a complex history of interactions between males and females. In most species with internal fertilization, males transfer ejaculates that contain much more than sperm (6). This “cocktail” has been found to reduce competition from other males and induce females to produce eggs, sometimes at a cost to female Department of Ecology and Evolutionary Biology, and Institute of Marine Sciences, University of California Santa Cruz, CA, USA. E-mail: [email protected]
sciencemag.org SCIENCE
27 FEBRUARY 2015 • VOL 347 ISSUE 6225
Published by AAAS
PHOTO: SAM COTTON
exposure treatments (including a cocktail of three monoclonal antibodies and an approach based on RNA interference). Nearly all of the 65 or so antiviral drugs approved for clinical use in the United States are for treating chronic virus infections (including human immunodeficiency virus, hepatitis B virus, and hepatitis C virus). Drugs for more acute viral infections (such as influenza viruses, poxviruses, and herpesviruses) tend to be less effective overall, and translating data from cell culture studies into clinical use is challenging. As Ebola infection typically progresses quickly, the standard metric of the effective concentration values that reduce virus titers by 50% should probably be discarded for this virus. Reducing virus concentrations by 50% is highly unlikely to provide any clinical benefit; rather, aiming for a 90 or 99% reducwtion is more applicable for Ebola. Interestingly, tetrandrine functions in this range in vitro, but given its mode of action as a viral entry inhibitor, it may be better as a prophylactic than as a treatment unless used in combined therapy. Currently, there are over 60 compounds that have been suggested to be effective against Ebola and/or Marburg virus infections (10), but most of these compounds do not have a clear mechanism of action, whereas there is some such understanding for tetrandrine. A few of the compounds have advanced to clinical trials for Ebola (e.g., brincidofovir and favipiravir) largely in the absence of convincing preclinical data. As one may find ethical arguments to support such trials in the current situation, one needs to be concerned about lack of efficacy that may do more harm than good. Deriving improved versions of drugs such as tetrandrine may eventually lead to a therapeutic approach, but as it stands (the same hold true for other anti-Ebola compounds), that day is not just around the corner. ■
Delineating Ebola entry Darryl Falzarano and Heinz Feldmann Science 347, 947 (2015); DOI: 10.1126/science.aaa8121
This copy is for your personal, non-commercial use only.
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The following resources related to this article are available online at www.sciencemag.org (this information is current as of February 26, 2015 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/347/6225/947.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/347/6225/947.full.html#related This article cites 10 articles, 2 of which can be accessed free: http://www.sciencemag.org/content/347/6225/947.full.html#ref-list-1 This article appears in the following subject collections: Microbiology http://www.sciencemag.org/cgi/collection/microbio
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2015 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on February 26, 2015
Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here.
Delineating Ebola entry By Darryl Falzarano1 and Heinz Feldmann2
ILLUSTRATION: ADAPTED BY C. BICKEL/SCIENCE
T
tinic acid adenine dinucleotide phosphate (NAADP)–stimulated intracellular calcium channels—known as the TPCs. These channels are mainly localized to endosomes and lysosomes (acidic compartments where contents are degraded). Through the use of cells lacking TPC2, small interfering RNA, and small-molecule inhibitors, Sakurai et al. determined that TPC2 is required for Ebola entry. Moreover, the requirement for
he means by which Ebola virus enters a cell are becoming less mysterious. Although a definitive cell surface receptor for the virus, if there is one, remains to be identified, the mechanism of gaining entry is beginning to be fleshed out. Once inside the cell, the importance of numerous sequential processes is becoming better understood. On page 995 of this issue, Sakurai et al. (1) add another element to the viral entry pathway by showing that a calcium channel called two-pore channel 2 (TPC2) is required for Ebola virus release of the viral genome into the host cell. After Ebola’s surface glycoprotein binds to receptors, which may be nonspecific and possibly comprise numerous carbohydrate proteins (2), the virus enters the cell via macropinocytosis, a nonselective process of engulfment (3, 4) (see the figure). Once internalized into Endosome a membrane-bound vesicle (endosome), Ebola glycoproteins are cleaved (5) while being exposed to an increasingly acidic and reducing environment. All this occurs in the endosome where an essential interaction with a protein called Niemann-Pick C1 (NPC1) was proposed to result in the release of the virus’s Late endosome genetic material in a process known as fusion (6, 7). A small interfering RNA screen identified calcium signaling in the host cell, among other events, as necessary for Ebola virus entry (8). L-type calcium channels were NPC1 initially pinpointed as the key element involved. However, one of Lysosome? five compounds that block these channels did not prevent Ebola infection, suggesting that another mechanism is also involved. The effective inhibitors also block nico-
TPC2 was shown to be specific to the glycoprotein of Ebola virus, suggesting a highly specific endosomal processing pathway. Previous work demonstrated that NPC1 is also essential for Ebola virus entry and proposed that it may function as an endosomal receptor that triggers fusion (7). However, the findings of Sakurai et al. suggest that NPC1 is not likely required for fusion itself because late endosomes expressing TPC2, but not NPC1, correlated with productive infection in cultured cells. This indicates that interaction Receptor of the cleaved glycoprotein with (carbohydrate-binding protein) NPC1 occurs before the glycoprotein interaction with TPC2 and prior to fusion. Sakurai et al. further demonstrate the relevance of TPC2 by Macropinocytosis blocking the channel’s activity with the drug tetrandrine, which improved survival of Ebola-infected mice. This supports the important role of TPC2 for Ebola virus infection; however, it does not indicate that a viable treatment is close at hand. The partial Cellular effectiveness of the drug in mice protease was further reduced by delaying treatment by 1 day. This questions whether any protection would be observed in a macaque model (the “gold standard” for Ebola drug Tetrandrine efficacy testing) (9). Tetrandrine, (blocks infection) originally a traditional Chinese medicine (found in the plant Stephania tetrandra), is not apTPC2 proved for use in humans (except NAADP in China). In addition, the dose given to mice by Sakurai et al. was many times the half-maximal inhibitory dose observed in tissue 2+ Ca culture, and likely exceeds doses that would be considered safe for humans. Given the mode of action, it seems unlikely that tetranTPC2 drine treatment would be superior to the most advanced Ebola postCa2+
1
Vaccine and Infectious Disease Organization– International Vaccine Centre, University of Saskatchewan, Saskatoon, SK Canada. 2Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Hamilton, MT, USA. E-mail: [email protected]
Fusion Genome release Virus replication
SCIENCE sciencemag.org
Ebola’s entry. A model of infection shows that once the virus is internalized into an endosome, the viral glycoprotein is cleaved and binds to NPC1. The calcium channel TPC2 is then activated prior to a fusion event that releases the viral genome into the cell. The drug tetrandrine blocks TPC2. 27 FEBRUARY 2015 • VOL 347 ISSUE 6225
Published by AAAS
947
Downloaded from www.sciencemag.org on February 26, 2015
A specific calcium channel is required for Ebola virus to infect a cell
INSIGHTS | P E R S P E C T I V E S
REFERENCES
1. Y. Sakurai et al., Science 347, 995 (2015). 2. C. L. Hunt, N. J. Lennemann, W. Maury, Viruses 4, 258 (2012). 3. M. F. Saeed, A. A. Kolokoltsov, T. Albrecht, R. A. Davey, PLOS Pathog. 6, e1001110 (2010). 4. A. Nanbo et al., PLOS Pathog. 6, e1001121 (2010). 5. K. Chandran, N. J. Sullivan, U. Felbor, S. P. Whelan, J. M. Cunningham, Science 308, 1643 (2005). 6. J. E. Carette et al., Nature 477, 340 (2011). 7. M. Côté et al., Nature 477, 344 (2011). 8. A. A. Kolokoltsov, M. F. Saeed, A. N. Freiberg, M. R. Holbrook, R. A. Davey, Drug Dev. Res. 70, 255 (2009). 9. X. Qiu et al., Nature 514, 47 (2014). 10. E. Picazo, F. Giordanetto, Drug Discov. Today 10.1016/j. drudis.2014.12.010 (2014). ACKNOWLEDGMENTS
This work was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. 10.1126/science.aaa8121
948
Female avoids male harassment Male produces steroid hormone 20E and forms a mating plug
Female receives 20E, which interacts with the MISO protein to stimulate egg production and decrease remating
Male fathers more ofspring
Male
Reduced immune response allows plasmodium infection Female
Coevolved sexual traits in male and female mosquitoes. Mitchell et al. show that as anophelene mosquitoes evolved, so have the traits that affect interactions between the sexes, and likely also their capacity as vectors of human malaria.
EVOLUTION
An unexpected cost of sex Coevolution of male and female mosquitoes influences whether mosquitoes transmit human malaria By Suzanne H. Alonzo
S
election arising from interactions between the sexes is responsible for some of the most striking diversity on the planet. These interactions generate coevolutionary dynamics between males and females that have shaped traits such as the striking courtship displays of male birds and the less winsome mating appendages of some male insects (1). But research on sexual selection has relevance beyond understanding the weird sex lives of animals. For example, human disturbance of sexual selection can lead to the loss of native species (2) and sexually selected male harassment of females can increase a species’ risk of extinction (3). On page 985 of this issue, Mitchell et al. (4) show that sexual selection can also be relevant to human health. The authors report evidence of a coevolved suite of male and female sexual traits in the mosquitoes that transmit the vector responsible for human malaria (see the figure). These traits include the evolution of a “mating plug” that allows males to transfer not only sperm but also the steroid hormone 20-hydroxyecdysone (20E) to females. In the female’s reproductive tract, 20E sets off a cascade of processes that increase egg production and decrease the rate
at which females mate with other males. Yet, this only happens in species where these male and female traits have coevolved. Mitchell et al. also demonstrate rapid evolution of the mating-induced stimulator of oogenesis (MISO) protein, which is produced by females and interacts with 20E inside the female to increase egg production. In the main vector of malaria, Anopheles gambiae, proteins that stimulate egg production are known to decrease female innate immunity, which increases the susceptibility of female mosquitoes to infection by the plasmodium that causes malaria (5). It is not currently known why this suite of traits arose in some but not all mosquitoes and if their presence can be used to predict whether a species will transmit human malaria. Mosquitoes are not exceptional in having a suite of coevolved traits that suggest a complex history of interactions between males and females. In most species with internal fertilization, males transfer ejaculates that contain much more than sperm (6). This “cocktail” has been found to reduce competition from other males and induce females to produce eggs, sometimes at a cost to female Department of Ecology and Evolutionary Biology, and Institute of Marine Sciences, University of California Santa Cruz, CA, USA. E-mail: [email protected]
sciencemag.org SCIENCE
27 FEBRUARY 2015 • VOL 347 ISSUE 6225
Published by AAAS
PHOTO: SAM COTTON
exposure treatments (including a cocktail of three monoclonal antibodies and an approach based on RNA interference). Nearly all of the 65 or so antiviral drugs approved for clinical use in the United States are for treating chronic virus infections (including human immunodeficiency virus, hepatitis B virus, and hepatitis C virus). Drugs for more acute viral infections (such as influenza viruses, poxviruses, and herpesviruses) tend to be less effective overall, and translating data from cell culture studies into clinical use is challenging. As Ebola infection typically progresses quickly, the standard metric of the effective concentration values that reduce virus titers by 50% should probably be discarded for this virus. Reducing virus concentrations by 50% is highly unlikely to provide any clinical benefit; rather, aiming for a 90 or 99% reducwtion is more applicable for Ebola. Interestingly, tetrandrine functions in this range in vitro, but given its mode of action as a viral entry inhibitor, it may be better as a prophylactic than as a treatment unless used in combined therapy. Currently, there are over 60 compounds that have been suggested to be effective against Ebola and/or Marburg virus infections (10), but most of these compounds do not have a clear mechanism of action, whereas there is some such understanding for tetrandrine. A few of the compounds have advanced to clinical trials for Ebola (e.g., brincidofovir and favipiravir) largely in the absence of convincing preclinical data. As one may find ethical arguments to support such trials in the current situation, one needs to be concerned about lack of efficacy that may do more harm than good. Deriving improved versions of drugs such as tetrandrine may eventually lead to a therapeutic approach, but as it stands (the same hold true for other anti-Ebola compounds), that day is not just around the corner. ■
Delineating Ebola entry Darryl Falzarano and Heinz Feldmann Science 347, 947 (2015); DOI: 10.1126/science.aaa8121
This copy is for your personal, non-commercial use only.
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here.
The following resources related to this article are available online at www.sciencemag.org (this information is current as of February 26, 2015 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/347/6225/947.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/347/6225/947.full.html#related This article cites 10 articles, 2 of which can be accessed free: http://www.sciencemag.org/content/347/6225/947.full.html#ref-list-1 This article appears in the following subject collections: Microbiology http://www.sciencemag.org/cgi/collection/microbio
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2015 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on February 26, 2015
Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here.
