Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs


RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.


Vyšlo v časopise: Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs. PLoS Pathog 6(7): e32767. doi:10.1371/journal.ppat.1000996
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000996

Souhrn

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.


Zdroje

1. BaulcombeD

2004 RNA silencing in plants. Nature 431 356 363

2. DingSW

VoinnetO

2007 Antiviral Immunity Directed by Small RNAs. Cell 130 413 426

3. BoucheN

LauresserguesD

GasciolliV

VaucheretH

2006 An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. Embo J 25 3347 3356

4. DelerisA

Gallego-BartolomeJ

BaoJ

KasschauKD

CarringtonJC

2006 Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313 68 71

5. MoissiardG

VoinnetO

2006 RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci U S A 103 19593 19598

6. VaucheretH

2008 Plant ARGONAUTES. Trends Plant Sci 13 350 358

7. HutvagnerG

SimardMJ

2008 Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol 9 22 32

8. MorelJB

GodonC

MourrainP

BeclinC

BoutetS

2002 Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14 629 639

9. QuF

YeX

MorrisTJ

2008 Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proc Natl Acad Sci U S A 105 14732 14737

10. ZhangX

YuanYR

PeiY

LinSS

TuschlT

2006 Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev 20 3255 3268

11. BaumbergerN

BaulcombeDC

2005 Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci U S A 102 11928 11933

12. BrodersenP

Sakvarelidze-AchardL

Bruun-RasmussenM

DunoyerP

YamamotoYY

2008 Widespread translational inhibition by plant miRNAs and siRNAs. Science 320 1185 1190

13. QiX

BaoFS

XieZ

2009 Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis. PLoS ONE 4 e4971

14. QuF

YeX

HouG

SatoS

ClementeTE

2005 RDR6 has a broad-spectrum but temperature-dependent antiviral defense role in Nicotiana benthamiana. J Virol 79 15209 15217

15. SchwachF

VaistijFE

JonesL

BaulcombeDC

2005 An RNA-dependent RNA polymerase prevents meristem invasion by potato virus X and is required for the activity but not the production of a systemic silencing signal. Plant Physiol 138 1842 1852

16. VoinnetO

2008 Use, tolerance and avoidance of amplified RNA silencing by plants. Trends Plant Sci 13 317 328

17. DonaireL

WangY

Gonzalez-IbeasD

MayerKF

ArandaMA

2009 Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology

18. WangXB

WuQ

ItoT

CilloF

LiWX

RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana. Proc Natl Acad Sci U S A 107 484 489

19. BurgyanJ

2008 Role of Silencing Suppressor Proteins. Methods Mol Biol 451 69 79

20. CsorbaT

PantaleoV

BurgyanJ

2009 RNA silencing: an antiviral mechanism. Adv Virus Res 75 35 71

21. ChapmanEJ

ProkhnevskyAI

GopinathK

DoljaVV

CarringtonJC

2004 Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18 1179 1186

22. LakatosL

CsorbaT

PantaleoV

ChapmanEJ

CarringtonJC

2006 Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. Embo J 25 2768 2780

23. HaasG

AzevedoJ

MoissiardG

GeldreichA

HimberC

2008 Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4. EMBO J 27 2102 2112

24. PazhouhandehM

DieterleM

MarroccoK

LechnerE

BerryB

2006 F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function. Proc Natl Acad Sci U S A 103 1994 1999

25. BaumbergerN

TsaiCH

LieM

HaveckerE

BaulcombeDC

2007 The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation. Curr Biol 17 1609 1614

26. Urcuqui-InchimaS

HaenniAL

BernardiF

2001 Potyvirus proteins: a wealth of functions. Virus Res 74 157 175

27. AnandalakshmiR

PrussGJ

GeX

MaratheR

MalloryAC

1998 A viral suppressor of gene silencing in plants. Proc Natl Acad Sci U S A 95 13079 13084

28. BrignetiG

VoinnetO

LiWX

JiLH

DingSW

1998 Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. Embo J 17 6739 6746

29. KasschauKD

CarringtonJC

1998 A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95 461 470

30. ValliA

Martin-HernandezAM

Lopez-MoyaJJ

GarciaJA

2006 RNA silencing suppression by a second copy of the P1 serine protease of Cucumber vein yellowing ipomovirus, a member of the family Potyviridae that lacks the cysteine protease HCPro. J Virol 80 10055 10063

31. LiW

HilfME

WebbSE

BakerCA

AdkinsS

2008 Presence of P1b and absence of HC-Pro in Squash vein yellowing virus suggests a general feature of the genus Ipomovirus in the family Potyviridae. Virus Res 135 213 219

32. ValliA

DujovnyG

GarciaJA

2008 Protease activity, self interaction, and small interfering RNA binding of the silencing suppressor p1b from cucumber vein yellowing ipomovirus. J Virol 82 974 986

33. ColinetD

KummertJ

LepoivreP

1998 The nucleotide sequence and genome organization of the whitefly transmitted sweetpotato mild mottle virus: a close relationship with members of the family Potyviridae. Virus Res 53 187 196

34. TillS

LejeuneE

ThermannR

BortfeldM

HothornM

2007 A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain. Nat Struct Mol Biol 14 897 903

35. TillS

LadurnerAG

2007 RNA Pol IV plays catch with Argonaute 4. Cell 131 643 645

36. AdamsMJ

AntoniwJF

FauquetCM

2005 Molecular criteria for genus and species discrimination within the family Potyviridae. Arch Virol 150 459 479

37. ShibolethYM

HaronskyE

LeibmanD

AraziT

WasseneggerM

2007 The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. J Virol 81 13135 13148

38. LichnerZ

SilhavyD

BurgyanJ

2003 Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences. J Gen Virol 84 975 980

39. HuangY

JiL

HuangQ

VassylyevDG

ChenX

2009 Structural insights into mechanisms of the small RNA methyltransferase HEN1. Nature 461 823 827

40. RamachandranV

ChenX

2008 Small RNA metabolism in Arabidopsis. Trends Plant Sci 13 368 374

41. LozsaR

CsorbaT

LakatosL

BurgyanJ

2008 Inhibition of 3′ modification of small RNAs in virus-infected plants require spatial and temporal co-expression of small RNAs and viral silencing-suppressor proteins. Nucleic Acids Res 36 4099 4107

42. MatrangaC

TomariY

ShinC

BartelDP

ZamorePD

2005 Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123 607 620

43. PantaleoV

SzittyaG

BurgyanJ

2007 Molecular Bases of Viral RNA Targeting by Viral Small Interfering RNA-Programmed RISC. J Virol 81 3797 3806

44. ParizottoEA

DunoyerP

RahmN

HimberC

VoinnetO

2004 In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev 18 2237 2242

45. SzittyaG

MolnarA

SilhavyD

HornyikC

BurgyanJ

2002 Short defective interfering RNAs of tombusviruses are not targeted but trigger post-transcriptional gene silencing against their helper virus. Plant Cell 14 359 372

46. SilhavyD

MolnarA

LucioliA

SzittyaG

HornyikC

2002 A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. Embo J 21 3070 3080

47. HockJ

WeinmannL

EnderC

RudelS

KremmerE

2007 Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells. EMBO Rep 8 1052 1060

48. PhamJW

PellinoJL

LeeYS

CarthewRW

SontheimerEJ

2004 A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117 83 94

49. CsorbaT

LozsaR

HutvagnerG

BurgyanJ

Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1. Plant J

50. OmarovRT

CiomperlikJJ

ScholthofHB

2007 RNAi-associated ssRNA-specific ribonucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete siRNA-containing effector complex. Proc Natl Acad Sci U S A 104 1714 1719

51. El-ShamiM

PontierD

LahmyS

BraunL

PicartC

2007 Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes Dev 21 2539 2544

52. QiY

DenliAM

HannonGJ

2005 Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 19 421 428

53. BortolamiolD

PazhouhandehM

MarroccoK

GenschikP

Ziegler-GraffV

2007 The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing. Curr Biol 17 1615 1621

54. JanssenD

MartinG

VelascoL

GomezP

SegundoE

2005 Absence of a coding region for the helper component-proteinase in the genome of cucumber vein yellowing virus, a whitefly-transmitted member of the Potyviridae. Arch Virol 150 1439 1447

55. MbanzibwaDR

TianY

MukasaSB

ValkonenJP

2009 Cassava brown streak virus (Potyviridae) encodes a putative Maf/HAM1 pyrophosphatase implicated in reduction of mutations and a P1 proteinase that suppresses RNA silencing but contains no HC-Pro. J Virol 83 6934 6940

56. ChenHY

YangJ

LinC

YuanYA

2008 Structural basis for RNA-silencing suppression by Tomato aspermy virus protein 2b. EMBO Rep 9 754 760

57. Diaz-PendonJA

LiF

LiWX

DingSW

2007 Suppression of antiviral silencing by cucumber mosaic virus 2b protein in Arabidopsis is associated with drastically reduced accumulation of three classes of viral small interfering RNAs. Plant Cell 19 2053 2063

58. KarlowskiWM

ZielezinskiA

CarrereJ

PontierD

LagrangeT

Genome-wide computational identification of WG/GW Argonaute-binding proteins in Arabidopsis. Nucleic Acids Res

59. Behm-AnsmantI

RehwinkelJ

DoerksT

StarkA

BorkP

2006 mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev 20 1885 1898

60. EulalioA

HuntzingerE

IzaurraldeE

2008 GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay. Nat Struct Mol Biol 15 346 353

61. LiuJ

RivasFV

WohlschlegelJ

YatesJR3rd

ParkerR

2005 A role for the P-body component GW182 in microRNA function. Nat Cell Biol 7 1261 1266

62. BuhlerM

VerdelA

MoazedD

2006 Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125 873 886

63. VerdelA

JiaS

GerberS

SugiyamaT

GygiS

2004 RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303 672 676

64. Bies-EtheveN

PontierD

LahmyS

PicartC

VegaD

2009 RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor family. EMBO Rep 10 649 654

65. HeXJ

HsuYF

ZhuS

WierzbickiAT

PontesO

2009 An effector of RNA-directed DNA methylation in arabidopsis is an ARGONAUTE 4- and RNA-binding protein. Cell 137 498 508

66. LazzarettiD

TournierI

IzaurraldeE

2009 The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence bound transcripts independently of Argonaute proteins. RNA 15 1059 1066

67. BhattacharjeeS

ZamoraA

AzharMT

SaccoMA

LambertLH

2009 Virus resistance induced by NB-LRR proteins involves Argonaute4-dependent translational control. Plant J 58 940 951

68. ZamorePD

2004 Plant RNAi: How a viral silencing suppressor inactivates siRNA. Curr Biol 14 R198 200

69. CuellarWJ

KreuzeJF

RajamakiML

CruzadoKR

UntiverosM

2009 Elimination of antiviral defense by viral RNase III. Proc Natl Acad Sci U S A 106 10354 10358

70. KreuzeJF

SavenkovEI

CuellarW

LiX

ValkonenJP

2005 Viral class 1 RNase III involved in suppression of RNA silencing. J Virol 79 7227 7238

71. MukasaSB

RubaihayoPR

ValkonenJPT

2006 Interactions between a crinivirus, an ipomovirus and a potyvirus in coinfected sweetpotato plants. Plant Pathology 55 458 467

72. KerenyiZ

MeraiZ

HiripiL

BenkovicsA

GyulaP

2008 Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay. EMBO J 27 1585 1595

73. LakatosL

SzittyaG

SilhavyD

BurgyanJ

2004 Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. Embo J 23 876 884

74. ValocziA

HornyikC

VargaN

BurgyanJ

KauppinenS

2004 Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32 e175

75. ValliA

Lopez-MoyaJJ

GarciaJA

2007 Recombination and gene duplication in the evolutionary diversification of P1 proteins in the family Potyviridae. J Gen Virol 88 1016 1028

76. SakaiJ

MoriM

MorishitaT

TanakaM

HanadaK

1997 Complete nucleotide sequence and genome organization of sweet potato feathery mottle virus (S strain) genomic RNA: the large coding region of the P1 gene. Arch Virol 142 1553 1562

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