Influenza Virus M2 Protein Mediates ESCRT-Independent Membrane Scission

Many viruses utilize host ESCRT proteins for budding; however, influenza virus budding is thought to be ESCRT-independent. In this study we have found a role for the influenza virus M2 proton-selective ion channel protein in mediating virus budding. We observed that a highly conserved amphipathic helix located within the M2 cytoplasmic tail mediates a cholesterol-dependent alteration in membrane curvature. The 17 amino acid amphipathic helix is sufficient for budding into giant unilamellar vesicles, and mutation of this sequence inhibited budding of transfected M2 protein in vivo. We show that M2 localizes to the neck of budding virions and that mutation of the M2 amphipathic helix results in failure of the virus to undergo membrane scission and virion release. These data suggest that M2 mediates the final steps of budding for influenza viruses, bypassing the need for host ESCRT proteins.

[1]  S. Goff,et al.  Infectivity of Moloney Murine Leukemia Virus Defective in Late Assembly Events Is Restored by Late Assembly Domains of Other Retroviruses , 2000, Journal of Virology.

[2]  R. Lamb,et al.  Influenza virus assembly and budding in raft-derived microdomains: a quantitative analysis of the surface distribution of HA, NA and M2 proteins. , 2005, Virology.

[3]  D. Lingwood,et al.  Order of lipid phases in model and plasma membranes , 2009, Proceedings of the National Academy of Sciences.

[4]  Yukiko Muramoto,et al.  The Cytoplasmic Tail of the Influenza A Virus M2 Protein Plays a Role in Viral Assembly , 2006, Journal of Virology.

[5]  J. Molotkovsky,et al.  Cholesterol modulates interaction between an amphipathic class A peptide, Ac-18A-NH2, and phosphatidylcholine bilayers. , 2002, Biochemistry.

[6]  Deborah A. Brown,et al.  Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface , 1992, Cell.

[7]  A. Pekosz,et al.  Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production , 2006, Journal of Virology.

[8]  Andrew Pekosz,et al.  The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging , 2005, Journal of Virology.

[9]  R. Lamb,et al.  Controlling influenza virus replication by inhibiting its proton channel. , 2007, Molecular bioSystems.

[10]  J. Chou,et al.  Structure and mechanism of the M2 proton channel of influenza A virus , 2008, Nature.

[11]  R. Lamb,et al.  Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion , 2001, The EMBO journal.

[12]  Jeremy G. Carlton,et al.  The ESCRT machinery: new functions in viral and cellular biology. , 2009, Biochemical Society transactions.

[13]  R. Lamb,et al.  The proton selective ion channels of influenza A and B viruses , 2005 .

[14]  P. Schwille,et al.  PI(4,5)P2 degradation promotes the formation of cytoskeleton-free model membrane systems. , 2009, Chemphyschem : a European journal of chemical physics and physical chemistry.

[15]  Y. Guan,et al.  Generation of recombinant influenza A virus without M2 ion-channel protein by introduction of a point mutation at the 5' end of the viral intron. , 2005, The Journal of general virology.

[16]  Kai Simons,et al.  Lipid rafts and signal transduction , 2000, Nature Reviews Molecular Cell Biology.

[17]  R. Lamb,et al.  A functionally defined model for the M2 proton channel of influenza A virus suggests a mechanism for its ion selectivity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[18]  A Helenius,et al.  Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins , 1996, Journal of virology.

[19]  Berend Smit,et al.  Effect of cholesterol on the structure of a phospholipid bilayer , 2009, Proceedings of the National Academy of Sciences.

[20]  M. Ben Amar,et al.  Budding and fission of a multiphase vesicle , 2005, The European physical journal. E, Soft matter.

[21]  R. Lamb,et al.  The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway. , 1996, Molecular biology of the cell.

[22]  R. Lamb,et al.  Mechanisms for enveloped virus budding: can some viruses do without an ESCRT? , 2008, Virology.

[23]  R. Lamb,et al.  Reconstitution of the influenza virus M2 ion channel in lipid bilayers , 1994, The Journal of Membrane Biology.

[24]  P. Digard,et al.  The Rab11 Pathway Is Required for Influenza A Virus Budding and Filament Formation , 2010, Journal of Virology.

[25]  G. Trugnan,et al.  Non-Metabolic Membrane Tubulation and Permeability Induced by Bioactive Peptides , 2007, PloS one.

[26]  Alok K. Chakrabarti,et al.  Role of Transmembrane Domain and Cytoplasmic Tail Amino Acid Sequences of Influenza A Virus Neuraminidase in Raft Association and Virus Budding , 2004, Journal of Virology.

[27]  R. Lamb,et al.  Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape , 1997, The EMBO journal.

[28]  P. Bassereau,et al.  Integrin reconstituted in GUVs: a biomimetic system to study initial steps of cell spreading. , 2009, Biochimica et biophysica acta.

[29]  Jennifer Lippincott-Schwartz,et al.  Membrane scission by the ESCRT-III complex , 2009, Nature.

[30]  R. Lamb,et al.  Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers , 2003, Protein science : a publication of the Protein Society.

[31]  R. Lamb,et al.  Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel , 2009, Proceedings of the National Academy of Sciences.

[32]  R. Epand,et al.  Effect of end group blockage on the properties of a class A amphipathic helical peptide , 1993, Proteins.

[33]  P. Digard,et al.  Budding of filamentous and non-filamentous influenza A virus occurs via a VPS4 and VPS28-independent pathway. , 2009, Virology.

[34]  R. Lamb,et al.  The influenza A virus spliced messenger RNA M mRNA3 is not required for viral replication in tissue culture. , 2008, The Journal of general virology.

[35]  K. Gaus,et al.  Actin Dynamics Drive Membrane Reorganization and Scission in Clathrin-Independent Endocytosis , 2010, Cell.

[36]  Charles J. Russell,et al.  Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  P. Gregory,et al.  February , 1890, The Hospital.

[38]  Cinque S. Soto,et al.  pH-induced conformational change of the influenza M2 protein C-terminal domain. , 2008, Biochemistry.

[39]  R. Lamb,et al.  Influenza Virus Hemagglutinin (H3 Subtype) Requires Palmitoylation of Its Cytoplasmic Tail for Assembly: M1 Proteins of Two Subtypes Differ in Their Ability To Support Assembly , 2005, Journal of Virology.

[40]  R. Lamb,et al.  Influenza Virus Hemagglutinin and Neuraminidase, but Not the Matrix Protein, Are Required for Assembly and Budding of Plasmid-Derived Virus-Like Particles , 2007, Journal of Virology.

[41]  B. Antonny Membrane deformation by protein coats. , 2006, Current opinion in cell biology.

[42]  D. Nayak,et al.  Formation of influenza virus particles lacking hemagglutinin on the viral envelope , 1986, Journal of virology.

[43]  R. Lamb,et al.  Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions , 1988, Journal of virology.

[44]  G. Drin,et al.  Amphipathic helices and membrane curvature , 2010, FEBS letters.

[45]  Kai Simons,et al.  Plasma membranes are poised for activation of raft phase coalescence at physiological temperature , 2008, Proceedings of the National Academy of Sciences.

[46]  G. Oster,et al.  Endocytic vesicle scission by lipid phase boundary forces , 2006, Proceedings of the National Academy of Sciences.

[47]  D. Gerlier,et al.  Virus Entry, Assembly, Budding, and Membrane Rafts , 2003, Microbiology and Molecular Biology Reviews.

[48]  R. Lamb,et al.  Influenza Virus M2 Ion Channel Protein Is Necessary for Filamentous Virion Formation , 2010, Journal of Virology.

[49]  C. Dempsey The actions of melittin on membranes. , 1990, Biochimica et biophysica acta.

[50]  M. Kozlov,et al.  Lipids in biological membrane fusion , 1995, The Journal of Membrane Biology.

[51]  E. Möncke-Buchner,et al.  The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein , 2005, European Biophysics Journal.

[52]  Patricia Bassereau,et al.  A new method for the reconstitution of membrane proteins into giant unilamellar vesicles. , 2004, Biophysical journal.

[53]  R. Lamb,et al.  The M2 Proton Channels of Influenza A and B Viruses* , 2006, Journal of Biological Chemistry.

[54]  E. Medcalf,et al.  Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions , 2007, The Journal of general virology.

[55]  R. Lamb,et al.  The Influenza Virus M2 Protein Cytoplasmic Tail Interacts with the M1 Protein and Influences Virus Assembly at the Site of Virus Budding , 2008, Journal of Virology.

[56]  R. Lamb,et al.  Characterization of the membrane association of the influenza virus matrix protein in living cells. , 1996, Virology.