Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin

The hemagglutinin (HA) of influenza virus is a type I transmembrane glycoprotein which is acylated with long-chain fatty acids. In this study we have used oligonucleotide-directed mutagenesis of cloned cDNA and a simian virus 40 expression system to determine the fatty acid binding site in HA and to examine possible functions of covalently linked fatty acids. The results show that the HA is acylated through thioester linkages at three highly conserved cysteine residues located in the cytoplasmic domain and at the carboxy-terminal end of the transmembrane region, whereas a cysteine located in the middle of the membrane-spanning domain is not acylated. Mutants lacking fatty acids at individual or all three attachment sites acquire endoglycosidase H-resistant oligosaccharide side chains, are cleaved into HA1 and HA2 subunits, and are transported to the plasma membrane at rates similar to that of wild-type HA. All mutants are membrane bound and not secreted into the medium. These results exclude transport signal and membrane-anchoring functions of covalently linked fatty acids for this integral membrane glycoprotein. Furthermore, lack of acylation has no obvious influence on the biological activities of HA: cells expressing fatty acid-free HA bind to and, after brief exposure to mildly acidic pH, fuse with erythrocytes; the HA-induced polykaryon formation is not impaired, either. Other possible functions of covalently linked fatty acids in integral membrane glycoproteins which cannot be examined in conventional cDNA expression systems are discussed.

[1]  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.

[2]  H. Klenk,et al.  The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different fatty acids. , 1990, Virology.

[3]  P. Palese,et al.  Introduction of site-specific mutations into the genome of influenza virus. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Rothman,et al.  Fatty acyl-coenzyme a is required for budding of transport vesicles from Golgi cisternae , 1989, Cell.

[5]  D. Marc,et al.  Role of myristoylation of poliovirus capsid protein VP4 as determined by site‐directed mutagenesis of its N‐terminal sequence. , 1989, The EMBO journal.

[6]  J. Sodroski,et al.  Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[7]  S. Doyle,et al.  Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus. , 1989, The Journal of biological chemistry.

[8]  R. Grand,et al.  Acylation of viral and eukaryotic proteins. , 1989, The Biochemical journal.

[9]  H. Klenk,et al.  The Molecular Biology of Influenza Virus Pathogenicity , 1988, Advances in Virus Research.

[10]  M. Schmidt,et al.  Chemical identification of cysteine as palmitoylation site in a transmembrane protein (Semliki Forest virus E1). , 1988, The Journal of biological chemistry.

[11]  H. Feldmann,et al.  The structure of serotype H10 hemagglutinin of influenza A virus: comparison of an apathogenic avian and a mammalian strain pathogenic for mink. , 1988, Virology.

[12]  A. S. Bogachuk,et al.  Two adjacent cysteine residues in the C‐terminal cytoplasmic fragment of bovine rhodopsin are palmitylated , 1988, FEBS letters.

[13]  I. Trowbridge,et al.  Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid‐attachment site. , 1987, The EMBO journal.

[14]  H. Klenk,et al.  Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus. , 1986, The EMBO journal.

[15]  R. Webster,et al.  Assembly of influenza hemagglutinin trimers and its role in intracellular transport , 1986, The Journal of cell biology.

[16]  J. Sambrook,et al.  Expression of wild-type and mutant forms of influenza hemagglutinin: The role of folding in intracellular transport , 1986, Cell.

[17]  Michael F. G. Schmidt,et al.  Membrane fusion induced by influenza virus hemagglutinin requires protein bound fatty acids , 1986, FEBS letters.

[18]  G. Hämmerling,et al.  The HLA-D-associated invariant chain binds palmitic acid at the cysteine adjacent to the membrane segment. , 1986, The Journal of biological chemistry.

[19]  T. Shioda,et al.  Determination of the complete nucleotide sequence of the Sendai virus genome RNA and the predicted amino acid sequences of the F, HN and L proteins. , 1986, Nucleic acids research.

[20]  F. Eckstein,et al.  The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. , 1985, Nucleic acids research.

[21]  B. Lambrecht,et al.  On the structure of the acyl linkage and the function of fatty acyl chains in the influenza virus haemagglutinin and the glycoproteins of Semliki Forest virus. , 1985, The Journal of general virology.

[22]  H. Klenk,et al.  Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two‐dimensional 1H n.m.r. and methylation analysis. , 1985, The EMBO journal.

[23]  J. Sambrook,et al.  Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport , 1985, The Journal of cell biology.

[24]  R. Lamb,et al.  Fusion protein of the paramyxovirus simian virus 5: nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Schmidt.  The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus. , 1984, The EMBO journal.

[26]  M. Schmidt,et al.  Cell-free fatty acid acylation of Semliki Forest viral polypeptides with microsomal membranes from eukaryotic cells. , 1984, The Journal of biological chemistry.

[27]  J. Rose,et al.  The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[28]  M. Gething,et al.  Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion , 1982, Nature.

[29]  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.

[30]  M. Schmidt,et al.  Acylation of virol. spike glycoproteins: A feature of enveloped RNA viruses , 1982, Virology.

[31]  H. Klenk,et al.  Influenza viruses cause hemolysis and fusion of cells. , 1981, Virology.

[32]  Y. Barenholz,et al.  Fluorescence anisotropy of a fatty acid covalently linked in vivo to the glycoprotein of vesicular stomatitis virus. , 1981, The Journal of biological chemistry.

[33]  J. Skehel,et al.  Disulphide bonds of haemagglutinin of Asian influenza virus , 1981, Nature.

[34]  Harvey F. Lodish,et al.  Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein , 1980, Cell.

[35]  M. Schmidt,et al.  Relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and Sindbis virus membrane glycoproteins. , 1980, The Journal of biological chemistry.

[36]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[37]  P. Choppin,et al.  Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. , 1975, Virology.

[38]  R. Lamb.  Genes and Proteins of the Influenza Viruses , 1989 .

[39]  M. Veit,et al.  Different palmitoylation of paramyxovirus glycoproteins. , 1989, Virology.

[40]  J. Gordon,et al.  The biology and enzymology of eukaryotic protein acylation. , 1988, Annual review of biochemistry.

[41]  A. Schultz,et al.  Fatty acylation of proteins. , 1988, Annual review of cell biology.

[42]  J. Skehel,et al.  The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. , 1987, Annual review of biochemistry.

[43]  F. Cross,et al.  An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins , 1985, Nature.

[44]  W. Stoffel,et al.  The primary structure of bovine brain myelin lipophilin (proteolipid apoprotein). , 1983, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.