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

ABSTRACT The influenza A virus hemagglutinin (HA) transmembrane domain boundary region and the cytoplasmic tail contain three cysteines (residues 555, 562, and 565 for the H3 HA subtype) that are highly conserved among the 16 HA subtypes and which are each modified by the covalent addition of palmitic acid. Previous analysis of the role of these conserved cysteine residues led to differing data, suggesting either no role for HA palmitoylation or an important role for HA palmitoylation. To reexamine the role of these residues in the influenza virus life cycle, a series of cysteine-to-serine mutations were introduced into the HA gene of influenza virus A/Udorn/72 (Ud) (H3N2) by using a highly efficient reverse genetics system. Mutant viruses containing HA-C562S and HA-C565S mutations had reduced growth and failed to form plaques in MDCK cells but formed wild-type-like plaques in an MDCK cell line expressing wild-type HA. In cell-cell fusion assays, nonpalmitoylated H3 HA, in both cDNA-transfected and virus-infected cells, was fully competent for HA-mediated membrane fusion. When the HA cytoplasmic tail cysteine mutants were examined for lipid raft association, using as the criterion Triton X-100 insolubility, loss of raft association did not show a direct correlation with a reduction in virus replication. However, mutant virus assembly was reduced in parallel with reduced virus replication. Additionally, a reassortant of strain A/WSN/33 (WSN), containing the Ud HA gene with mutations C555S, C562S, and C565S, produced virus that could form plaques on regular MDCK cells and had only moderately decreased replication, suggesting differences in the interactions between Ud and WSN HA and internal viral proteins. Analysis of M1 mutants containing substitutions in the six residues that differ between the Ud and WSN M1 proteins indicated that a constellation of residues are responsible for the difference between the M1 proteins in their ability to support virus assembly with nonpalmitoylated H3 HA.

[1]  H. Klenk,et al.  Acylation-Mediated Membrane Anchoring of Avian Influenza Virus Hemagglutinin Is Essential for Fusion Pore Formation and Virus Infectivity , 2005, Journal of Virology.

[2]  R. Kuhn,et al.  Association of sindbis virus capsid protein with phospholipid membranes and the E2 glycoprotein: implications for alphavirus assembly. , 2005, Biochemistry.

[3]  Subrata Barman,et al.  Assembly and budding of influenza virus , 2004, Virus Research.

[4]  W. Bornmann,et al.  B Cell Signaling Is Regulated by Induced Palmitoylation of CD81* , 2004, Journal of Biological Chemistry.

[5]  J. Bhattacharya,et al.  Human Immunodeficiency Virus Type 1 Envelope Glycoproteins That Lack Cytoplasmic Domain Cysteines: Impact on Association with Membrane Lipid Rafts and Incorporation onto Budding Virus Particles , 2004, Journal of Virology.

[6]  W. Barclay,et al.  The M1 matrix protein controls the filamentous phenotype of influenza A virus. , 2004, Virology.

[7]  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.

[8]  A. García-Sastre,et al.  Reverse genetics studies on the filamentous morphology of influenza A virus. , 2003, The Journal of general virology.

[9]  Z. Ye,et al.  Restriction of Viral Replication by Mutation of the Influenza Virus Matrix Protein , 2002, Journal of Virology.

[10]  T. Sakai,et al.  Fatty Acids on the A/USSR/77 Influenza Virus Hemagglutinin Facilitate the Transition from Hemifusion to Fusion Pore Formation , 2002, Journal of Virology.

[11]  R. Webster,et al.  Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses , 2002, Journal of Virology.

[12]  R. Lamb,et al.  Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture , 2002, Journal of Virology.

[13]  E. Freed,et al.  Plasma membrane rafts play a critical role in HIV-1 assembly and release , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[14]  F. Baudin,et al.  Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer. , 2001, Virology.

[15]  P. S. Kim,et al.  Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[16]  T. Harder,et al.  Selective Accumulation of Raft-Associated Membrane Protein Lat in T Cell Receptor Signaling Assemblies , 2000, The Journal of cell biology.

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

[18]  Ayub Ali,et al.  Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein , 2000, Journal of Virology.

[19]  Andrew Pekosz,et al.  Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins , 2000, Journal of Virology.

[20]  R. Lamb,et al.  The cytoplasmic tails of the influenza virus spike glycoproteins are required for normal genome packaging. , 2000, Virology.

[21]  R. Ruigrok,et al.  Membrane interaction of influenza virus M1 protein. , 2000, Virology.

[22]  H. Klenk,et al.  Caspase-Dependent N-Terminal Cleavage of Influenza Virus Nucleocapsid Protein in Infected Cells , 1999, Journal of Virology.

[23]  Tokiko Watanabe,et al.  Generation of influenza A viruses entirely from cloned cDNAs. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M. Roth,et al.  Role of Lipid Modifications in Targeting Proteins to Detergent-resistant Membrane Rafts , 1999, The Journal of Biological Chemistry.

[25]  P. Scheiffele,et al.  Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane* , 1999, The Journal of Biological Chemistry.

[26]  H. Klenk,et al.  Acylation of the influenza hemagglutinin modulates fusion activity. , 1998, Virology.

[27]  R. Lamb,et al.  The M1 and M2 proteins of influenza A virus are important determinants in filamentous particle formation. , 1998, Virology.

[28]  R. Lamb,et al.  The role of the cytoplasmic tail region of influenza virus hemagglutinin in formation and growth of fusion pores. , 1997, Virology.

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

[30]  M. Luo,et al.  Structure of a bifunctional membrane-RNA binding protein, influenza virus matrix protein M1 , 1997, Nature Structural Biology.

[31]  M Enami,et al.  Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein , 1996, Journal of virology.

[32]  R. Lamb,et al.  Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity , 1996, Journal of Virology.

[33]  R. Lamb,et al.  Effects of antibody to the influenza A virus M2 protein on M2 surface expression and virus assembly. , 1995, Virology.

[34]  M. Veit,et al.  Assessment of fusogenic properties of influenza virus hemagglutinin deacylated by site-directed mutagenesis and hydroxylamine treatment. , 1995, Virology.

[35]  A. Ward,et al.  Specific changes in the M1 protein during adaptation of influenza virus to mouse , 1995, Archives of Virology.

[36]  S. Ley,et al.  Palmitoylation of multiple Src-family kinases at a homologous N-terminal motif. , 1994, The Biochemical journal.

[37]  P. Palese,et al.  Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation , 1994, Journal of virology.

[38]  M. Schlesinger,et al.  Site-directed mutations in the Sindbis virus E2 glycoprotein identify palmitoylation sites and affect virus budding , 1993, Journal of virology.

[39]  M. Roth,et al.  Effects of altering palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin , 1992, Journal of virology.

[40]  R. Lamb,et al.  Alterations to influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity , 1992, Journal of virology.

[41]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[42]  H. Niwa,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector. , 1991, Gene.

[43]  J. Skehel,et al.  Deacylation of the hemagglutinin of influenza A/Aichi/2/68 has no effect on membrane fusion properties. , 1991, Virology.

[44]  M. Roth,et al.  A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin , 1991, The Journal of cell biology.

[45]  Y Tateno,et al.  Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. , 1991, Virology.

[46]  H. Klenk,et al.  Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin , 1991, Journal of virology.

[47]  C. Naeve,et al.  Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membrane fusion. , 1990, The EMBO journal.

[48]  J. Parvin,et al.  Amplification, expression, and packaging of a foreign gene by influenza virus , 1989, Cell.

[49]  J. Thorner,et al.  A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae , 1989, Cell.

[50]  J. Zimmerberg,et al.  Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing , 1989, The Journal of cell biology.

[51]  R. Lamb,et al.  Growth restriction of influenza A virus by M2 protein antibody is genetically linked to the M1 protein. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[52]  M. Schlesinger,et al.  Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation. , 1982, The Journal of biological chemistry.

[53]  R. Lamb,et al.  Identification of a second protein (M2) encoded by RNA segment 7 of influenza virus. , 1981, Virology.

[54]  A. B. Will,et al.  Lipid Raft Microdomains: A Gateway for Compartmentalized Trafficking of Ebola and , 2002 .

[55]  R. Lamb,et al.  Orthomyxoviridae: The Viruses and Their Replication. , 1996 .