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. 1999 Jun 1;18(11):3164–3172. doi: 10.1093/emboj/18.11.3164

Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity.

T Ahola 1, A Lampio 1, P Auvinen 1, L Kääriäinen 1
PMCID: PMC1171397  PMID: 10357827

Abstract

The replication complexes of all positive strand RNA viruses of eukaryotes are associated with membranes. In the case of Semliki Forest virus (SFV), the main determinant of membrane attachment seems to be the virus-encoded non-structural protein NSP1, the capping enzyme of the viral mRNAs, which has guanine-7-methyltransferase and guanylyltransferase activities. We show here that both enzymatic activities of SFV NSP1 are inactivated by detergents and reactivated by anionic phospholipids, especially phosphatidylserine. The region of NSP1 responsible for binding to membranes as well as to liposomes was mapped to a short segment, which is conserved in the large alphavirus-like superfamily of viruses. A synthetic peptide of 20 amino acids from the putative binding site competed with in vitro synthesized NSP1 for binding to liposomes containing phosphatidylserine. These findings suggest a molecular mechanism by which RNA virus replicases attach to intracellular membranes and why they depend on the membranous environment.

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Selected References

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  1. Ahola T., Käriäinen L. Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):507–511. doi: 10.1073/pnas.92.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahola T., Laakkonen P., Vihinen H., Käriäinen L. Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities. J Virol. 1997 Jan;71(1):392–397. doi: 10.1128/jvi.71.1.392-397.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ames G. F. Lipids of Salmonella typhimurium and Escherichia coli: structure and metabolism. J Bacteriol. 1968 Mar;95(3):833–843. doi: 10.1128/jb.95.3.833-843.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borhani D. W., Rogers D. P., Engler J. A., Brouillette C. G. Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation. Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12291–12296. doi: 10.1073/pnas.94.23.12291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Buck K. W. Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res. 1996;47:159–251. doi: 10.1016/S0065-3527(08)60736-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cornell R. B. Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 1. Negative surface charge dependence for activation. Biochemistry. 1991 Jun 18;30(24):5873–5880. doi: 10.1021/bi00238a010. [DOI] [PubMed] [Google Scholar]
  8. Dunne S. J., Cornell R. B., Johnson J. E., Glover N. R., Tracey A. S. Structure of the membrane binding domain of CTP:phosphocholine cytidylyltransferase. Biochemistry. 1996 Sep 17;35(37):11975–11984. doi: 10.1021/bi960821+. [DOI] [PubMed] [Google Scholar]
  9. Echeverri A. C., Dasgupta A. Amino terminal regions of poliovirus 2C protein mediate membrane binding. Virology. 1995 Apr 20;208(2):540–553. doi: 10.1006/viro.1995.1185. [DOI] [PubMed] [Google Scholar]
  10. Eidelman O., Blumenthal R., Walter A. Composition of octyl glucoside-phosphatidylcholine mixed micelles. Biochemistry. 1988 Apr 19;27(8):2839–2846. doi: 10.1021/bi00408a027. [DOI] [PubMed] [Google Scholar]
  11. Foster P. A., Fulcher C. A., Houghten R. A., Zimmerman T. S. Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine. Blood. 1990 May 15;75(10):1999–2004. [PubMed] [Google Scholar]
  12. Froshauer S., Kartenbeck J., Helenius A. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol. 1988 Dec;107(6 Pt 1):2075–2086. doi: 10.1083/jcb.107.6.2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gilbert G. E., Baleja J. D. Membrane-binding peptide from the C2 domain of factor VIII forms an amphipathic structure as determined by NMR spectroscopy. Biochemistry. 1995 Mar 7;34(9):3022–3031. doi: 10.1021/bi00009a033. [DOI] [PubMed] [Google Scholar]
  14. Gilbert G. E., Furie B. C., Furie B. Binding of human factor VIII to phospholipid vesicles. J Biol Chem. 1990 Jan 15;265(2):815–822. [PubMed] [Google Scholar]
  15. Goldbach R. Genome similarities between plant and animal RNA viruses. Microbiol Sci. 1987 Jul;4(7):197–202. [PubMed] [Google Scholar]
  16. Hardy W. R., Strauss J. H. Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans. J Virol. 1989 Nov;63(11):4653–4664. doi: 10.1128/jvi.63.11.4653-4664.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hjelmeland L. M., Chrambach A. Solubilization of functional membrane proteins. Methods Enzymol. 1984;104:305–318. doi: 10.1016/s0076-6879(84)04097-0. [DOI] [PubMed] [Google Scholar]
  18. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  19. Jacobs B. L., Langland J. O. When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology. 1996 May 15;219(2):339–349. doi: 10.1006/viro.1996.0259. [DOI] [PubMed] [Google Scholar]
  20. Koonin E. V., Dolja V. V. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol. 1993;28(5):375–430. doi: 10.3109/10409239309078440. [DOI] [PubMed] [Google Scholar]
  21. LaStarza M. W., Lemm J. A., Rice C. M. Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis. J Virol. 1994 Sep;68(9):5781–5791. doi: 10.1128/jvi.68.9.5781-5791.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laakkonen P., Ahola T., Käriäinen L. The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme. J Biol Chem. 1996 Nov 8;271(45):28567–28571. doi: 10.1074/jbc.271.45.28567. [DOI] [PubMed] [Google Scholar]
  23. Laakkonen P., Hyvönen M., Peränen J., Käriäinen L. Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli. J Virol. 1994 Nov;68(11):7418–7425. doi: 10.1128/jvi.68.11.7418-7425.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lama J., Paul A. V., Harris K. S., Wimmer E. Properties of purified recombinant poliovirus protein 3aB as substrate for viral proteinases and as co-factor for RNA polymerase 3Dpol. J Biol Chem. 1994 Jan 7;269(1):66–70. [PubMed] [Google Scholar]
  25. Lampio A., Siissalo I., Gahmberg C. G. Oxidation of glycolipids in liposomes by galactose oxidase. Eur J Biochem. 1988 Dec 1;178(1):87–91. doi: 10.1111/j.1432-1033.1988.tb14432.x. [DOI] [PubMed] [Google Scholar]
  26. Lee J. Y., Marshall J. A., Bowden D. S. Characterization of rubella virus replication complexes using antibodies to double-stranded RNA. Virology. 1994 Apr;200(1):307–312. doi: 10.1006/viro.1994.1192. [DOI] [PubMed] [Google Scholar]
  27. Peränen J., Käriäinen L. Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected BHK cells. J Virol. 1991 Mar;65(3):1623–1627. doi: 10.1128/jvi.65.3.1623-1627.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Peränen J., Laakkonen P., Hyvönen M., Käriäinen L. The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles. Virology. 1995 Apr 20;208(2):610–620. doi: 10.1006/viro.1995.1192. [DOI] [PubMed] [Google Scholar]
  29. Peränen J., Rikkonen M., Hyvönen M., Käriäinen L. T7 vectors with modified T7lac promoter for expression of proteins in Escherichia coli. Anal Biochem. 1996 May 1;236(2):371–373. doi: 10.1006/abio.1996.0187. [DOI] [PubMed] [Google Scholar]
  30. Peränen J., Rikkonen M., Liljeström P., Käriäinen L. Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2. J Virol. 1990 May;64(5):1888–1896. doi: 10.1128/jvi.64.5.1888-1896.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Peränen J., Takkinen K., Kalkkinen N., Käriäinen L. Semliki Forest virus-specific non-structural protein nsP3 is a phosphoprotein. J Gen Virol. 1988 Sep;69(Pt 9):2165–2178. doi: 10.1099/0022-1317-69-9-2165. [DOI] [PubMed] [Google Scholar]
  32. Picot D., Loll P. J., Garavito R. M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature. 1994 Jan 20;367(6460):243–249. doi: 10.1038/367243a0. [DOI] [PubMed] [Google Scholar]
  33. Rigaud J. L., Mosser G., Lacapere J. J., Olofsson A., Levy D., Ranck J. L. Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct Biol. 1997 Apr;118(3):226–235. doi: 10.1006/jsbi.1997.3848. [DOI] [PubMed] [Google Scholar]
  34. Rikkonen M., Peränen J., Käriäinen L. ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2. J Virol. 1994 Sep;68(9):5804–5810. doi: 10.1128/jvi.68.9.5804-5810.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rouser G., Fkeischer S., Yamamoto A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970 May;5(5):494–496. doi: 10.1007/BF02531316. [DOI] [PubMed] [Google Scholar]
  36. Rozanov M. N., Koonin E. V., Gorbalenya A. E. Conservation of the putative methyltransferase domain: a hallmark of the 'Sindbis-like' supergroup of positive-strand RNA viruses. J Gen Virol. 1992 Aug;73(Pt 8):2129–2134. doi: 10.1099/0022-1317-73-8-2129. [DOI] [PubMed] [Google Scholar]
  37. Sawicki D. L., Sawicki S. G., Keränen S., Käriäinen L. Specific Sindbis virus-coded function for minus-strand RNA synthesis. J Virol. 1981 Aug;39(2):348–358. doi: 10.1128/jvi.39.2.348-358.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Takkinen K. Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus. Nucleic Acids Res. 1986 Jul 25;14(14):5667–5682. doi: 10.1093/nar/14.14.5667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Teterina N. L., Gorbalenya A. E., Egger D., Bienz K., Ehrenfeld E. Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells. J Virol. 1997 Dec;71(12):8962–8972. doi: 10.1128/jvi.71.12.8962-8972.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tokuyasu K. T. Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy. Histochem J. 1989 Mar;21(3):163–171. doi: 10.1007/BF01007491. [DOI] [PubMed] [Google Scholar]
  42. Towner J. S., Ho T. V., Semler B. L. Determinants of membrane association for poliovirus protein 3AB. J Biol Chem. 1996 Oct 25;271(43):26810–26818. doi: 10.1074/jbc.271.43.26810. [DOI] [PubMed] [Google Scholar]
  43. Wang H. L., O'Rear J., Stollar V. Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity. Virology. 1996 Mar 15;217(2):527–531. doi: 10.1006/viro.1996.0147. [DOI] [PubMed] [Google Scholar]
  44. Wang Y. F., Sawicki S. G., Sawicki D. L. Sindbis virus nsP1 functions in negative-strand RNA synthesis. J Virol. 1991 Feb;65(2):985–988. doi: 10.1128/jvi.65.2.985-988.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wu S. X., Ahlquist P., Kaesberg P. Active complete in vitro replication of nodavirus RNA requires glycerophospholipid. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11136–11140. doi: 10.1073/pnas.89.23.11136. [DOI] [PMC free article] [PubMed] [Google Scholar]

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