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PLoS Pathog. Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway
Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway (PLoS Pathogens, abstract, edited)
[Source: PLoS Pathogens, full text: <cite cite="http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1001329?utm_so urce=feedburner&utm_medium=feed&utm_campaign=Feed% 3A+plospathogens%2FNewArticles+%28Ambra+-+Pathogens+New+Articles%29">PLoS Pathogens: Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway</cite>. Abstract, edited.]
Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway
Erik de Vries 1, Donna M. Tscherne 2, Marleen J. Wienholts 1, Viviana Cobos-Jim?nez 1, Florine Scholte 1, Adolfo Garc?a-Sastre 2,3,4, Peter J. M. Rottier 1, Cornelis A. M. de Haan 1*
1 Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands,
2 Department of Microbiology, Mount Sinai School of Medicine, New York, United States of America,
3 Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, United States of America,
4 Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, United States of America
Abstract
Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis.
Author Summary
Attachment to and entry into a host cell are the first crucial steps in establishing a successful virus infection and critical factors in determining host cell and species tropism. Influenza A virus (IAV) attaches to host cells by binding of its major surface protein, hemagglutinin, to sialic acids that are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. IAV subsequently enters cells of birds and a wide variety of mammals via receptor-mediated endocytosis using clathrin as well as via (an) alternative uncharacterized route(s). The elucidation of the endocytic pathways taken by IAV has been hampered by their apparent redundancy in establishing a productive infection. By manipulating the entry conditions we have established experimental settings that allow the separate analysis of dynamin-dependent (including clathrin-mediated endocytosis) and independent entry of IAV. Collectively, our results indicate macropinocytosis, the main route for the non-selective uptake of extracellular fluid by cells, as an alternative IAV entry route. As the dynamin-dependent and -independent IAV entry routes are redundant and independent, their separate manipulation was crucial for the identification and characterization of the alternative IAV entry route. A similar strategy might be applicable to the study of endocytic pathways taken by other viruses.
Citation: de Vries E, Tscherne DM, Wienholts MJ, Cobos-Jim?nez V, Scholte F, et al. (2011) Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway. PLoS Pathog 7(3): e1001329. doi:10.1371/journal.ppat.1001329
Editor: Andrew Pekosz, Johns Hopkins University BSPH, United States of America
Received: August 13, 2010; Accepted: March 2, 2011; Published: March 31, 2011
Copyright: ? 2011 de Vries et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been financially supported by the program: ?Impulse Veterinary Avian Influenza Research? in the Netherlands. This work was partially supported by National Institute of Health grant U01 AI1074539 and by CRIP (HHSN266200700010C), an NIAID Center of Excellence for Influenza Research and Surveillance (to A.G-S.). D.M.T. is supported by NIH fellowship 1F32AI081428. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: c.a.m.dehaan@uu.nl
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[Source: PLoS Pathogens, full text: <cite cite="http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1001329?utm_so urce=feedburner&utm_medium=feed&utm_campaign=Feed% 3A+plospathogens%2FNewArticles+%28Ambra+-+Pathogens+New+Articles%29">PLoS Pathogens: Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway</cite>. Abstract, edited.]
Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway
Erik de Vries 1, Donna M. Tscherne 2, Marleen J. Wienholts 1, Viviana Cobos-Jim?nez 1, Florine Scholte 1, Adolfo Garc?a-Sastre 2,3,4, Peter J. M. Rottier 1, Cornelis A. M. de Haan 1*
1 Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands,
2 Department of Microbiology, Mount Sinai School of Medicine, New York, United States of America,
3 Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, United States of America,
4 Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, United States of America
Abstract
Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis.
Author Summary
Attachment to and entry into a host cell are the first crucial steps in establishing a successful virus infection and critical factors in determining host cell and species tropism. Influenza A virus (IAV) attaches to host cells by binding of its major surface protein, hemagglutinin, to sialic acids that are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. IAV subsequently enters cells of birds and a wide variety of mammals via receptor-mediated endocytosis using clathrin as well as via (an) alternative uncharacterized route(s). The elucidation of the endocytic pathways taken by IAV has been hampered by their apparent redundancy in establishing a productive infection. By manipulating the entry conditions we have established experimental settings that allow the separate analysis of dynamin-dependent (including clathrin-mediated endocytosis) and independent entry of IAV. Collectively, our results indicate macropinocytosis, the main route for the non-selective uptake of extracellular fluid by cells, as an alternative IAV entry route. As the dynamin-dependent and -independent IAV entry routes are redundant and independent, their separate manipulation was crucial for the identification and characterization of the alternative IAV entry route. A similar strategy might be applicable to the study of endocytic pathways taken by other viruses.
Citation: de Vries E, Tscherne DM, Wienholts MJ, Cobos-Jim?nez V, Scholte F, et al. (2011) Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway. PLoS Pathog 7(3): e1001329. doi:10.1371/journal.ppat.1001329
Editor: Andrew Pekosz, Johns Hopkins University BSPH, United States of America
Received: August 13, 2010; Accepted: March 2, 2011; Published: March 31, 2011
Copyright: ? 2011 de Vries et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been financially supported by the program: ?Impulse Veterinary Avian Influenza Research? in the Netherlands. This work was partially supported by National Institute of Health grant U01 AI1074539 and by CRIP (HHSN266200700010C), an NIAID Center of Excellence for Influenza Research and Surveillance (to A.G-S.). D.M.T. is supported by NIH fellowship 1F32AI081428. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: c.a.m.dehaan@uu.nl
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