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. 1990 Dec;9(12):3857–3866. doi: 10.1002/j.1460-2075.1990.tb07604.x

Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membrane fusion.

C W Naeve 1, D Williams 1
PMCID: PMC552153  PMID: 2249653

Abstract

The covalent attachment of fatty acid moieties to proteins is a widespread post-translational modification of viral and cell proteins yet the functional consequences of acylation are not well understood. We have determined that the A/Japan/305/57 influenza virus hemagglutinin (HA) contains three potential acylation sites at cysteine residues 211, 218 and 221 in the cytoplasmic domain of the molecule. Site-directed mutagenesis of one or more of these sites has no effect on biosynthesis, transport or receptor binding activity of the molecule; however, modification of any single site is sufficient to abolish completely or inhibit severely membrane fusion activity, a function essential for virus infectivity. We present a molecular model of the transmembrane and cytoplasmic domains of the HA to illustrate the potential orientation of these fatty acids and to provide a conceptual framework for further experimentation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arumugham R. G., Seid R. C., Jr, Doyle S., Hildreth S. W., Paradiso P. R. Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus. J Biol Chem. 1989 Jun 25;264(18):10339–10342. [PubMed] [Google Scholar]
  2. Bathurst I. C., Chester N., Gibson H. L., Dennis A. F., Steimer K. S., Barr P. J. N myristylation of the human immunodeficiency virus type 1 gag polyprotein precursor in Saccharomyces cerevisiae. J Virol. 1989 Jul;63(7):3176–3179. doi: 10.1128/jvi.63.7.3176-3179.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buss J. E., Sefton B. M. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog. J Virol. 1985 Jan;53(1):7–12. doi: 10.1128/jvi.53.1.7-12.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  7. Chow M., Newman J. F., Filman D., Hogle J. M., Rowlands D. J., Brown F. Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature. 1987 Jun 11;327(6122):482–486. doi: 10.1038/327482a0. [DOI] [PubMed] [Google Scholar]
  8. Cohen C., Parry D. A. Alpha-helical coiled coils and bundles: how to design an alpha-helical protein. Proteins. 1990;7(1):1–15. doi: 10.1002/prot.340070102. [DOI] [PubMed] [Google Scholar]
  9. Copeland C. S., Doms R. W., Bolzau E. M., Webster R. G., Helenius A. Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J Cell Biol. 1986 Oct;103(4):1179–1191. doi: 10.1083/jcb.103.4.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deisenhofer J., Michel H. The Photosynthetic Reaction Center from the Purple Bacterium Rhodopseudomonas viridis. Science. 1989 Sep 29;245(4925):1463–1473. doi: 10.1126/science.245.4925.1463. [DOI] [PubMed] [Google Scholar]
  11. Deléage G., Roux B. An algorithm for protein secondary structure prediction based on class prediction. Protein Eng. 1987 Aug-Sep;1(4):289–294. doi: 10.1093/protein/1.4.289. [DOI] [PubMed] [Google Scholar]
  12. Doyle C., Roth M. G., Sambrook J., Gething M. J. Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport. J Cell Biol. 1985 Mar;100(3):704–714. doi: 10.1083/jcb.100.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  14. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  15. Gascuel O., Golmard J. L. A simple method for predicting the secondary structure of globular proteins: implications and accuracy. Comput Appl Biosci. 1988 Aug;4(3):357–365. doi: 10.1093/bioinformatics/4.3.357. [DOI] [PubMed] [Google Scholar]
  16. Gething M. J., Bye J., Skehel J., Waterfield M. Cloning and DNA sequence of double-stranded copies of haemagglutinin genes from H2 and H3 strains elucidates antigenic shift and drift in human influenza virus. Nature. 1980 Sep 25;287(5780):301–306. doi: 10.1038/287301a0. [DOI] [PubMed] [Google Scholar]
  17. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  18. Gething M. J., Sambrook J. Cell-surface expression of influenza haemagglutinin from a cloned DNA copy of the RNA gene. Nature. 1981 Oct 22;293(5834):620–625. doi: 10.1038/293620a0. [DOI] [PubMed] [Google Scholar]
  19. Gething M. J., Sambrook J. Construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein. Nature. 1982 Dec 16;300(5893):598–603. doi: 10.1038/300598a0. [DOI] [PubMed] [Google Scholar]
  20. Gutierrez L., Magee A. I., Marshall C. J., Hancock J. F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. doi: 10.1002/j.1460-2075.1989.tb03478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  22. Harlow E., Pim D. C., Crawford L. V. Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells. J Virol. 1981 Feb;37(2):564–573. doi: 10.1128/jvi.37.2.564-573.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hedo J. A., Collier E., Watkinson A. Myristyl and palmityl acylation of the insulin receptor. J Biol Chem. 1987 Jan 25;262(3):954–957. [PubMed] [Google Scholar]
  24. Heuckeroth R. O., Gordon J. I. Altered membrane association of p60v-src and a murine 63-kDa N-myristoyl protein after incorporation of an oxygen-substituted analog of myristic acid. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5262–5266. doi: 10.1073/pnas.86.14.5262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Huylebroeck D., Maertens G., Verhoeyen M., Lopez C., Raeymakers A., Jou W. M., Fiers W. High-level transient expression of influenza virus proteins from a series of SV40 late and early replacement vectors. Gene. 1988 Jun 30;66(2):163–181. doi: 10.1016/0378-1119(88)90354-x. [DOI] [PubMed] [Google Scholar]
  26. Jackson J. H., Cochrane C. G., Bourne J. R., Solski P. A., Buss J. E., Der C. J. Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3042–3046. doi: 10.1073/pnas.87.8.3042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jacobs E., Gheysen D., Thines D., Francotte M., de Wilde M. The HIV-1 Gag precursor Pr55gag synthesized in yeast is myristoylated and targeted to the plasma membrane. Gene. 1989 Jun 30;79(1):71–81. doi: 10.1016/0378-1119(89)90093-0. [DOI] [PubMed] [Google Scholar]
  28. Jørgensen E. C., Kjeldgaard N. O., Pedersen F. S., Jørgensen P. A nucleotide substitution in the gag N terminus of the endogenous ecotropic DBA/2 virus prevents Pr65gag myristylation and virus replication. J Virol. 1988 Sep;62(9):3217–3223. doi: 10.1128/jvi.62.9.3217-3223.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kaufman J. F., Krangel M. S., Strominger J. L. Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds. J Biol Chem. 1984 Jun 10;259(11):7230–7238. [PubMed] [Google Scholar]
  30. Kawaoka Y., Naeve C. W., Webster R. G. Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology. 1984 Dec;139(2):303–316. doi: 10.1016/0042-6822(84)90376-3. [DOI] [PubMed] [Google Scholar]
  31. Klein P., Kanehisa M., DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
  32. Kotwal G. J., Ghosh H. P. Role of fatty acid acylation of membrane glycoproteins. Absence of palmitic acid in glycoproteins of two serotypes of vesicular stomatitis virus. J Biol Chem. 1984 Apr 25;259(8):4699–4701. [PubMed] [Google Scholar]
  33. Kräusslich H. G., Hölscher C., Reuer Q., Harber J., Wimmer E. Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity. J Virol. 1990 May;64(5):2433–2436. doi: 10.1128/jvi.64.5.2433-2436.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  35. Lambrecht B., Schmidt M. F. Membrane fusion induced by influenza virus hemagglutinin requires protein bound fatty acids. FEBS Lett. 1986 Jun 23;202(1):127–132. doi: 10.1016/0014-5793(86)80662-7. [DOI] [PubMed] [Google Scholar]
  36. Levitt M. Conformational preferences of amino acids in globular proteins. Biochemistry. 1978 Oct 3;17(20):4277–4285. doi: 10.1021/bi00613a026. [DOI] [PubMed] [Google Scholar]
  37. Mack D., Kruppa J. Fatty acid acylation at the single cysteine residue in the cytoplasmic domain of the glycoprotein of vesicular-stomatitis virus. Biochem J. 1988 Dec 15;256(3):1021–1027. doi: 10.1042/bj2561021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Maxfield F. R., Scheraga H. A. Status of empirical methods for the prediction of protein backbone topography. Biochemistry. 1976 Nov 16;15(23):5138–5153. doi: 10.1021/bi00668a030. [DOI] [PubMed] [Google Scholar]
  39. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  40. Mohana Rao J. K., Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta. 1986 Jan 30;869(2):197–214. doi: 10.1016/0167-4838(86)90295-5. [DOI] [PubMed] [Google Scholar]
  41. Muszbek L., Laposata M. Covalent modification of platelet proteins by palmitate. Blood. 1989 Sep;74(4):1339–1347. [PubMed] [Google Scholar]
  42. Muszbek L., Laposata M. Glycoprotein Ib and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages. J Biol Chem. 1989 Jun 15;264(17):9716–9719. [PubMed] [Google Scholar]
  43. Novotný J., Auffray C. A program for prediction of protein secondary structure from nucleotide sequence data: application to histocompatibility antigens. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):243–255. doi: 10.1093/nar/12.1part1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Omary M. B., Trowbridge I. S. Covalent binding of fatty acid to the transferrin receptor in cultured human cells. J Biol Chem. 1981 May 25;256(10):4715–4718. [PubMed] [Google Scholar]
  45. Pellman D., Garber E. A., Cross F. R., Hanafusa H. An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins. 1985 Mar 28-Apr 3Nature. 314(6009):374–377. doi: 10.1038/314374a0. [DOI] [PubMed] [Google Scholar]
  46. Pillai S., Baltimore D. Myristoylation and the post-translational acquisition of hydrophobicity by the membrane immunoglobulin heavy-chain polypeptide in B lymphocytes. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7654–7658. doi: 10.1073/pnas.84.21.7654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Qian N., Sejnowski T. J. Predicting the secondary structure of globular proteins using neural network models. J Mol Biol. 1988 Aug 20;202(4):865–884. doi: 10.1016/0022-2836(88)90564-5. [DOI] [PubMed] [Google Scholar]
  48. Rein A., McClure M. R., Rice N. R., Luftig R. B., Schultz A. M. Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7246–7250. doi: 10.1073/pnas.83.19.7246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rose J. K., Adams G. A., Gallione C. J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2050–2054. doi: 10.1073/pnas.81.7.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Roth M. G., Compans R. W., Giusti L., Davis A. R., Nayak D. P., Gething M. J., Sambrook J. Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA. Cell. 1983 Jun;33(2):435–443. doi: 10.1016/0092-8674(83)90425-7. [DOI] [PubMed] [Google Scholar]
  51. Saermark T., Bex F. Acylation of HIV proteins. Biochem Soc Trans. 1989 Oct;17(5):869–871. doi: 10.1042/bst0170869. [DOI] [PubMed] [Google Scholar]
  52. Schmidt M. F. Acylation of viral spike glycoproteins: a feature of enveloped RNA viruses. Virology. 1982 Jan 15;116(1):327–338. doi: 10.1016/0042-6822(82)90424-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schmidt M. F., Lambrecht B. 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. J Gen Virol. 1985 Dec;66(Pt 12):2635–2647. doi: 10.1099/0022-1317-66-12-2635. [DOI] [PubMed] [Google Scholar]
  54. Schmidt M. F., Schlesinger M. J. Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein. Cell. 1979 Aug;17(4):813–819. doi: 10.1016/0092-8674(79)90321-0. [DOI] [PubMed] [Google Scholar]
  55. Schmidt M. F., Schlesinger M. J. Relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and Sindbis virus membrane glycoproteins. J Biol Chem. 1980 Apr 25;255(8):3334–3339. [PubMed] [Google Scholar]
  56. Schmidt M. F. The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus. EMBO J. 1984 Oct;3(10):2295–2300. doi: 10.1002/j.1460-2075.1984.tb02129.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schmidt M., Schmidt M. F., Rott R. Chemical identification of cysteine as palmitoylation site in a transmembrane protein (Semliki Forest virus E1). J Biol Chem. 1988 Dec 15;263(35):18635–18639. [PubMed] [Google Scholar]
  58. Schultz A. M., Henderson L. E., Oroszlan S. Fatty acylation of proteins. Annu Rev Cell Biol. 1988;4:611–647. doi: 10.1146/annurev.cb.04.110188.003143. [DOI] [PubMed] [Google Scholar]
  59. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  60. Schultz A. M., Rein A. Unmyristylated Moloney murine leukemia virus Pr65gag is excluded from virus assembly and maturation events. J Virol. 1989 May;63(5):2370–2373. doi: 10.1128/jvi.63.5.2370-2373.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Staufenbiel M. Fatty acids covalently bound to erythrocyte proteins undergo a differential turnover in vivo. J Biol Chem. 1988 Sep 25;263(27):13615–13622. [PubMed] [Google Scholar]
  62. Staufenbiel M. Fatty acylation of erythrocyte proteins. Biochem Soc Trans. 1989 Oct;17(5):863–864. doi: 10.1042/bst0170863. [DOI] [PubMed] [Google Scholar]
  63. Stegmann T., Doms R. W., Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. doi: 10.1146/annurev.bb.18.060189.001155. [DOI] [PubMed] [Google Scholar]
  64. Streuli C. H., Griffin B. E. Myristic acid is coupled to a structural protein of polyoma virus and SV40. Nature. 1987 Apr 9;326(6113):619–622. doi: 10.1038/326619a0. [DOI] [PubMed] [Google Scholar]
  65. Towler D. A., Gordon J. I., Adams S. P., Glaser L. The biology and enzymology of eukaryotic protein acylation. Annu Rev Biochem. 1988;57:69–99. doi: 10.1146/annurev.bi.57.070188.000441. [DOI] [PubMed] [Google Scholar]
  66. White J., Helenius A., Gething M. J. Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion. Nature. 1982 Dec 16;300(5893):658–659. doi: 10.1038/300658a0. [DOI] [PubMed] [Google Scholar]
  67. Wiley D. C., Skehel J. J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem. 1987;56:365–394. doi: 10.1146/annurev.bi.56.070187.002053. [DOI] [PubMed] [Google Scholar]
  68. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]
  69. Zdebska E., Antoniewicz J., Kościelak J. Characterization and quantitation of fatty acids covalently bound to erythrocyte membrane proteins: anion transporter contains 1 mol of fatty acid thiol ester. Arch Biochem Biophys. 1989 Aug 15;273(1):223–229. doi: 10.1016/0003-9861(89)90182-3. [DOI] [PubMed] [Google Scholar]

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