The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

[1]  The crystal structure of the influenza matrix protein M1 at neutral pH: M1-M1 protein interfaces can rotate in the oligomeric structures of M1. , 2001, Virology.

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

[3]  C. Benham,et al.  An influenza virus containing nine different RNA segments. , 1991, Virology.

[4]  A. García-Sastre,et al.  Chimeric Influenza A Viruses with a Functional Influenza B Virus Neuraminidase or Hemagglutinin , 2003, Journal of Virology.

[5]  G. K. Vorkunova,et al.  Influenza virus uncoating in infected cells and effect of rimantadine. , 1982, The Journal of general virology.

[6]  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.

[7]  L. Zhang,et al.  Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement , 2003, Journal of Clinical Virology.

[8]  A Helenius,et al.  Transport of incoming influenza virus nucleocapsids into the nucleus , 1991, Journal of virology.

[9]  R. Lamb,et al.  Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Palese,et al.  Determination of influenza virus proteins required for genome replication , 1990, Journal of virology.

[11]  O. Zhirnov.  Isolation of matrix protein M1 from influenza viruses by acid-dependent extraction with nonionic detergent. , 1992, Virology.

[12]  R. Lamb,et al.  Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. , 1991, Virology.

[13]  A. Davison,et al.  Molecular virology: a practical approach , 1993 .

[14]  R. Lamb,et al.  Permeation and Activation of the M2 Ion Channel of Influenza A Virus* , 2000, The Journal of Biological Chemistry.

[15]  J. Yewdell,et al.  Expression of influenza A virus internal antigens on the surface of infected P815 cells. , 1981, Journal of immunology.

[16]  P. Gómez-Puertas,et al.  Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins. , 1999, The Journal of general virology.

[17]  K. Ishii,et al.  Detection of ion channel activity in Xenopus laevis oocytes expressing Influenza C virus CM2 protein , 2003, Archives of Virology.

[18]  A. Hay,et al.  Influence of amantadine resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses. , 1992, Virology.

[19]  A. Hay,et al.  Regulation of pH by the M2 protein of influenza A viruses. , 1992, Virus research.

[20]  R. Lamb,et al.  Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal- anchor domain of a protein with an ectoplasmic NH2 terminus , 1988, The Journal of cell biology.

[21]  Shinji Watanabe,et al.  Influenza A Virus Can Undergo Multiple Cycles of Replication without M2 Ion Channel Activity , 2001, Journal of Virology.

[22]  K. Martin,et al.  Nuclear transport of influenza virus ribonucleoproteins: The viral matrix protein (M1) promotes export and inhibits import , 1991, Cell.

[23]  O. Weisz,et al.  Influenza Virus M2 Protein Slows Traffic along the Secretory Pathway , 1998, The Journal of Biological Chemistry.

[24]  J. Galarza,et al.  Formation of Wild-Type and Chimeric Influenza Virus-Like Particles following Simultaneous Expression of Only Four Structural Proteins , 2001, Journal of Virology.

[25]  A. Hay,et al.  Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells. , 1996, The Journal of physiology.

[26]  Hideo Goto,et al.  Selective incorporation of influenza virus RNA segments into virions , 2003, Proceedings of the National Academy of Sciences of the United States of America.

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

[28]  T. Parslow,et al.  Evidence for Segment-Nonspecific Packaging of the Influenza A Virus Genome , 2002, Journal of Virology.

[29]  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.

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

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

[32]  W. Barclay,et al.  Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity. , 2004, Virology.

[33]  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.

[34]  J. Skehel,et al.  The molecular basis of the specific anti‐influenza action of amantadine. , 1985, The EMBO journal.

[35]  R. Lamb,et al.  Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[36]  R. Lamb,et al.  Identification of a Membrane Targeting and Degradation Signal in the p42 Protein of Influenza C Virus , 2000, Journal of Virology.

[37]  M. Diamond,et al.  The utility of siRNA transcripts produced by RNA polymerase i in down regulating viral gene expression and replication of negative- and positive-strand RNA viruses. , 2003, Virology.

[38]  J. Skehel,et al.  On the mechanism of inhibition of influenza virus replication by amantadine hydrochloride. , 1978, The Journal of general virology.

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

[40]  R. Lamb,et al.  Effect of Cytoplasmic Tail Truncations on the Activity of the M2 Ion Channel of Influenza A Virus , 1999, Journal of Virology.

[41]  R. Lamb,et al.  Mechanism for Proton Conduction of the M2 Ion Channel of Influenza A Virus* , 2000, The Journal of Biological Chemistry.

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

[43]  A. Bukrinskaya,et al.  Uncoating of a rimantadine-resistant variant of influenza virus in the presence of rimantadine. , 1982, The Journal of general virology.

[44]  R. Lamb,et al.  Influenza B virus BM2 protein is an oligomeric integral membrane protein expressed at the cell surface. , 2003, Virology.

[45]  R. Lamb,et al.  The CM2 protein of influenza C virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins. , 1997, Virology.

[46]  Yoshihiro Kawaoka,et al.  Exploitation of Nucleic Acid Packaging Signals To Generate a Novel Influenza Virus-Based Vector Stably Expressing Two Foreign Genes , 2003, Journal of Virology.

[47]  R. Lamb,et al.  Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. , 1985, Cell.

[48]  O. Zhirnov.  Solubilization of matrix protein M1/M from virions occurs at different pH for orthomyxo- and paramyxoviruses. , 1990, Virology.

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

[50]  R. Lamb,et al.  Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport , 1994, Journal of virology.

[51]  H. Goto,et al.  Influenza B Virus Requires BM2 Protein for Replication , 2004, Journal of Virology.

[52]  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.

[53]  R. Lamb,et al.  Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block , 1993, Journal of virology.

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

[55]  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.

[56]  R. Lamb,et al.  The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus , 1996, The Journal of cell biology.

[57]  Tokiko Watanabe,et al.  Plasmid-Driven Formation of Influenza Virus-Like Particles , 2000, Journal of Virology.

[58]  R. Lamb,et al.  Characterization of the influenza virus M2 integral membrane protein and expression at the infected-cell surface from cloned cDNA , 1985, Journal of virology.

[59]  Shinji Watanabe,et al.  Influenza B Virus BM2 Protein Is Transported through the trans-Golgi Network as an Integral Membrane Protein , 2003, Journal of Virology.

[60]  R. Lamb,et al.  Nucleotide sequences of influenza A virus RNA segment 7: a comparison of five isolates. , 1989, Nucleic acids research.

[61]  R. Lamb,et al.  Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface , 1985, Cell.

[62]  H. Klenk,et al.  Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein , 1994, Journal of virology.

[63]  Lawrence H. Pinto,et al.  Influenza virus M2 protein has ion channel activity , 1992, Cell.

[64]  D. Rowlands,et al.  A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria. , 2004, The Journal of general virology.

[65]  P. Bayley,et al.  Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment. , 1992, Virology.

[66]  Shinji Watanabe,et al.  Influenza B Virus BM2 Protein Is a Crucial Component for Incorporation of Viral Ribonucleoprotein Complex into Virions during Virus Assembly , 2004, Journal of Virology.

[67]  R. Lamb,et al.  The molecular biology of influenza viruses and paramyxoviruses , 1993 .

[68]  P. Gómez-Puertas,et al.  Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding , 2000, Journal of Virology.

[69]  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.

[70]  R. Lamb,et al.  Influenza B virus BM2 protein has ion channel activity that conducts protons across membranes. , 2003, Developmental cell.

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

[72]  R. Ashley,et al.  The transmembrane domain of influenza A M2 protein forms amantadine-sensitive proton channels in planar lipid bilayers. , 1992, Virology.