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PMID: 17093195 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication.

Journal of virology ·Vol. 81 ·No. 2 ·2007-01-00 ·Pages 872-83

Spuul P, Salonen A, Merits A, Jokitalo E, Kääriäinen L, Ahola T

Abstract

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G245STLYTESRKLLRSWHLPSV264, has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.

MeSH Terms
Amino Acid Sequence Animals Cell Line Cell Membrane/metabolism,virology Cricetinae Green Fluorescent Proteins/genetics,metabolism HeLa Cells Humans Microscopy, Confocal Molecular Sequence Data Peptides/chemistry,metabolism Point Mutation RNA, Viral/biosynthesis RNA-Dependent RNA Polymerase/metabolism Recombinant Fusion Proteins/genetics,metabolism Semliki forest virus/genetics,metabolism,pathogenicity,physiology Viral Nonstructural Proteins/chemistry,genetics,metabolism Virus Replication
Chemicals
Peptides RNA, Viral Recombinant Fusion Proteins Viral Nonstructural Proteins Green Fluorescent Proteins RNA-Dependent RNA Polymerase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Spuul Pirjo
Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Salonen Anne
Merits Andres
Jokitalo Eija
Kääriäinen Leevi
Ahola Tero
References (50)
50 references, click to expand
  1. Global analysis of protein palmitoylation in yeast.
    Cell. 2006 Jun 2;125(5):1003-13 PMID: 16751107
  2. Functions of alphavirus nonstructural proteins in RNA replication.
    Prog Nucleic Acid Res Mol Biol. 2002;71:187-222 PMID: 12102555
  3. Effects of palmitoylation of replicase protein nsP1 on alphavirus infection.
    J Virol. 2000 Aug;74(15):6725-33 PMID: 10888610
  4. Mapping the lipid distribution in the membranes of BHK cells (mini-review).
    Mol Membr Biol. 1996 Apr-Jun;13(2):81-4 PMID: 8839451
  5. The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles.
    Virology. 1995 Apr 20;208(2):610-20 PMID: 7747433
  6. 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):2129-34 PMID: 1645151
  7. Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis.
    J Virol. 1994 Mar;68(3):1874-85 PMID: 8107248
  8. Studies of a putative amphipathic helix in the N-terminus of poliovirus protein 2C.
    Virology. 1994 Feb 15;199(1):188-99 PMID: 8116242
  9. Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis.
    EMBO J. 1994 Jun 15;13(12):2925-34 PMID: 7517863
  10. Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA.
    J Virol. 1994 Oct;68(10):6466-75 PMID: 8083984
  11. Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli.
    J Virol. 1994 Nov;68(11):7418-25 PMID: 7933125
  12. 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):507-11 PMID: 7831320
  13. Amino terminal regions of poliovirus 2C protein mediate membrane binding.
    Virology. 1995 Apr 20;208(2):540-53 PMID: 7747426
  14. Viral RNA replication in association with cellular membranes.
    Curr Top Microbiol Immunol. 2005;285:139-73 PMID: 15609503
  15. Subversion of cellular autophagosomal machinery by RNA viruses.
    PLoS Biol. 2005 May;3(5):e156 PMID: 15884975
  16. Testing the modularity of the N-terminal amphipathic helix conserved in picornavirus 2C proteins and hepatitis C NS5A protein.
    Virology. 2006 Jan 20;344(2):453-67 PMID: 16226781
  17. NMR structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5A from bovine viral diarrhea virus.
    Biochemistry. 2006 Feb 21;45(7):2221-33 PMID: 16475810
  18. A monotopic membrane protein goes solo.
    Structure. 2006 Apr;14(4):628-9 PMID: 16615901
  19. The crystal structure of carnitine palmitoyltransferase 2 and implications for diabetes treatment.
    Structure. 2006 Apr;14(4):713-23 PMID: 16615913
  20. Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion.
    J Virol. 2002 Oct;76(19):9856-67 PMID: 12208963
  21. The hepatitis C virus RNA-dependent RNA polymerase membrane insertion sequence is a transmembrane segment.
    J Virol. 2002 Dec;76(24):13088-93 PMID: 12438637
  22. Interaction of hepatitis C virus proteins with host cell membranes and lipids.
    Trends Cell Biol. 2002 Nov;12(11):517-23 PMID: 12446113
  23. Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.
    J Biol Chem. 2003 Jan 10;278(2):1012-21 PMID: 12244057
  24. Properly folded nonstructural polyprotein directs the semliki forest virus replication complex to the endosomal compartment.
    J Virol. 2003 Feb;77(3):1691-702 PMID: 12525603
  25. Topology of the membrane-associated hepatitis C virus protein NS4B.
    J Virol. 2003 May;77(9):5428-38 PMID: 12692244
  26. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage.
    J Mol Biol. 2003 Aug 29;331(5):991-1004 PMID: 12927536
  27. Regulation of the sequential processing of Semliki Forest virus replicase polyprotein.
    J Biol Chem. 2003 Oct 24;278(43):41636-45 PMID: 12917405
  28. A predicted amphipathic helix mediates plasma membrane localization of GRK5.
    J Biol Chem. 2004 Apr 23;279(17):17989-95 PMID: 14976207
  29. Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts.
    Virology. 2004 May 20;323(1):153-63 PMID: 15165827
  30. Isolation and basic characterization of temperature-sensitive mutants from Semliki Forest virus;.
    Acta Pathol Microbiol Scand B Microbiol Immunol. 1974 Dec;82(6):810-20 PMID: 4533222
  31. Semliki Forest virus-specific non-structural protein nsP3 is a phosphoprotein.
    J Gen Virol. 1988 Sep;69 ( Pt 9):2165-78 PMID: 2970523
  32. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes.
    J Cell Biol. 1988 Dec;107(6 Pt 1):2075-86 PMID: 2904446
  33. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release.
    J Virol. 1991 Aug;65(8):4107-13 PMID: 2072446
  34. Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli.
    Virology. 1991 Sep;184(1):423-7 PMID: 1831311
  35. Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA.
    Cell. 1992 Jan 10;68(1):119-31 PMID: 1310072
  36. Intensification of labelings of the immunogold silver staining method by gold toning.
    Brain Res Bull. 1992 Feb;28(2):343-5 PMID: 1375863
  37. Membrane binding mechanism of an RNA virus-capping enzyme.
    J Biol Chem. 2000 Dec 1;275(48):37853-9 PMID: 10984480
  38. Determinants of membrane association for poliovirus protein 3AB.
    J Biol Chem. 1996 Oct 25;271(43):26810-8 PMID: 8900162
  39. The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme.
    J Biol Chem. 1996 Nov 8;271(45):28567-71 PMID: 8910486
  40. Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells.
    J Virol. 1997 Dec;71(12):8962-72 PMID: 9371552
  41. Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line.
    Virology. 1997 Nov 24;238(2):198-211 PMID: 9400593
  42. ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex.
    J Virol. 1998 Aug;72(8):6689-98 PMID: 9658116
  43. Alphavirus replicase protein NSP1 induces filopodia and rearrangement of actin filaments.
    J Virol. 1998 Dec;72(12):10265-9 PMID: 9811773
  44. Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity.
    EMBO J. 1999 Jun 1;18(11):3164-72 PMID: 10357827
  45. Amphitropic proteins: regulation by reversible membrane interactions (review).
    Mol Membr Biol. 1999 Jul-Sep;16(3):217-35 PMID: 10503244
  46. Biogenesis of the Semliki Forest virus RNA replication complex.
    J Virol. 2001 Apr;75(8):3873-84 PMID: 11264376
  47. Mechanisms governing subcellular localization and function of human RGS2.
    J Biol Chem. 2001 Apr 27;276(17):14195-203 PMID: 11278586
  48. An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A.
    J Biol Chem. 2002 Mar 8;277(10):8130-9 PMID: 11744739
  49. RhoGDI-3 regulates RhoG and targets this protein to the Golgi complex through its unique N-terminal domain.
    Traffic. 2002 May;3(5):342-57 PMID: 11967128
  50. The role of the tethering proteins p115 and GM130 in transport through the Golgi apparatus in vivo.
    Mol Biol Cell. 2000 Feb;11(2):635-45 PMID: 10679020
Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.01785-06
Published
2007-01-00
Epub
2006-00-08
Pages
872-83
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC1797454
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.01785-06 PMC Full Text View journal details
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