The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex


The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.


Vyšlo v časopise: The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex. PLoS Pathog 6(6): e32767. doi:10.1371/journal.ppat.1000925
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000925

Souhrn

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.


Zdroje

1. PetroskiMD

DeshaiesRJ

2005 Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6 9 20

2. WillemsAR

SchwabM

TyersM

2004 A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta 1695 133 170

3. MahrourN

RedwineWB

FlorensL

SwansonSK

Martin-BrownS

2008 Characterization of Cullin-box sequences that direct recruitment of Cul2-Rbx1 and Cul5-Rbx2 modules to Elongin BC-based ubiquitin ligases. J Biol Chem 283 8005 8013

4. KamuraT

MaenakaK

KotoshibaS

MatsumotoM

KohdaD

2004 VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev 18 3055 3065

5. BullockAN

DebreczeniJE

EdwardsAM

SundstromM

KnappS

2006 Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proc Natl Acad Sci U S A 103 7637 7642

6. BullockAN

RodriguezMC

DebreczeniJE

SongyangZ

KnappS

2007 Structure of the SOCS4-ElonginB/C complex reveals a distinct SOCS box interface and the molecular basis for SOCS-dependent EGFR degradation. Structure 15 1493 1504

7. BabonJJ

SaboJK

SoetopoA

YaoS

BaileyMF

2008 The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase. J Mol Biol 381 928 940

8. MalimMH

EmermanM

2008 HIV-1 accessory proteins–ensuring viral survival in a hostile environment. Cell Host Microbe 3 388 398

9. YuX

YuY

LiuB

LuoK

KongW

2003 Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science 302 1056 1060

10. SheehyAM

GaddisNC

ChoiJD

MalimMH

2002 Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418 646 650

11. ChenG

HeZ

WangT

XuR

YuXF

2009 A patch of positively charged amino acids surrounding the human immunodeficiency virus type 1 Vif SLVx4Yx9Y motif influences its interaction with APOBEC3G. J Virol 83 8674 8682

12. HeZ

ZhangW

ChenG

XuR

YuXF

2008 Characterization of conserved motifs in HIV-1 Vif required for APOBEC3G and APOBEC3F interaction. J Mol Biol 381 1000 1011

13. RussellRA

PathakVK

2007 Identification of two distinct human immunodeficiency virus type 1 Vif determinants critical for interactions with human APOBEC3G and APOBEC3F. J Virol 81 8201 8210

14. MehleA

WilsonH

ZhangC

BrazierAJ

McPikeM

2007 Identification of an APOBEC3G binding site in human immunodeficiency virus type 1 Vif and inhibitors of Vif-APOBEC3G binding. J Virol 81 13235 13241

15. TianC

YuX

ZhangW

WangT

XuR

2006 Differential requirement for conserved tryptophans in human immunodeficiency virus type 1 Vif for the selective suppression of APOBEC3G and APOBEC3F. J Virol 80 3112 3115

16. XiaoZ

XiongY

ZhangW

TanL

EhrlichE

2007 Characterization of a novel Cullin5 binding domain in HIV-1 Vif. J Mol Biol 373 541 550

17. XiaoZ

EhrlichE

YuY

LuoK

WangT

2006 Assembly of HIV-1 Vif-Cul5 E3 ubiquitin ligase through a novel zinc-binding domain-stabilized hydrophobic interface in Vif. Virology 349 290 299

18. MehleA

ThomasER

RajendranKS

GabuzdaD

2006 A zinc-binding region in Vif binds Cul5 and determines cullin selection. J Biol Chem 281 17259 17265

19. LuoK

XiaoZ

EhrlichE

YuY

LiuB

2005 Primate lentiviral virion infectivity factors are substrate receptors that assemble with cullin 5-E3 ligase through a HCCH motif to suppress APOBEC3G. Proc Natl Acad Sci U S A 102 11444 11449

20. YuY

XiaoZ

EhrlichES

YuX

YuXF

2004 Selective assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and upstream cysteines. Genes Dev 18 2867 2872

21. SimonJH

SheehyAM

CarpenterEA

FouchierRA

MalimMH

1999 Mutational analysis of the human immunodeficiency virus type 1 Vif protein. J Virol 73 2675 2681

22. YangS

SunY

ZhangH

2001 The multimerization of human immunodeficiency virus type I Vif protein: a requirement for Vif function in the viral life cycle. J Biol Chem 276 4889 4893

23. DonahueJP

VetterML

MukhtarNA

D'AquilaRT

2008 The HIV-1 Vif PPLP motif is necessary for human APOBEC3G binding and degradation. Virology 377 49 53

24. KataropoulouA

BovolentaC

BelfioreA

TrabattiS

GarbelliA

2009 Mutational analysis of the HIV-1 auxiliary protein Vif identifies independent domains important for the physical and functional interaction with HIV-1 reverse transcriptase. Nucleic Acids Res 37 3660 3669

25. StanleyBJ

EhrlichES

ShortL

YuY

XiaoZ

2008 Structural insight into the human immunodeficiency virus Vif SOCS box and its role in human E3 ubiquitin ligase assembly. J Virol 82 8656 8663

26. BarraudP

PaillartJC

MarquetR

TisneC

2008 Advances in the structural understanding of Vif proteins. Curr HIV Res 6 91 99

27. ZhouP

LugovskoyAA

WagnerG

2001 A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins. J Biomol NMR 20 11 14

28. SchreiberG

2002 Kinetic studies of protein-protein interactions. Curr Opin Struct Biol 12 41 47

29. OkhrimenkoO

JelesarovI

2008 A survey of the year 2006 literature on applications of isothermal titration calorimetry. J Mol Recognit 21 1 19

30. SunZY

FruehDP

SelenkoP

HochJC

WagnerG

2005 Fast assignment of 15N-HSQC peaks using high-resolution 3D HNcocaNH experiments with non-uniform sampling. J Biomol NMR 33 43 50

31. DysonHJ

WrightPE

2002 Insights into the structure and dynamics of unfolded proteins from nuclear magnetic resonance. Adv Protein Chem 62 311 340

32. MarshJA

SinghVK

JiaZ

Forman-KayJD

2006 Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: implications for fibrillation. Protein Sci 15 2795 2804

33. TakeuchiK

NgE

MaliaTJ

WagnerG

2007 1-13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins. J Biomol NMR 38 89 98

34. CaseDA

2002 Molecular dynamics and NMR spin relaxation in proteins. Acc Chem Res 35 325 331

35. KnauthK

CartwrightE

FreundS

BycroftM

BuchbergerA

2009 VHL mutations linked to type 2C von Hippel-Lindau disease cause extensive structural perturbations in pVHL. J Biol Chem 284 10514 10522

36. FeldmanDE

ThulasiramanV

FerreyraRG

FrydmanJ

1999 Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC. Mol Cell 4 1051 1061

37. BrowerCS

ShilatifardA

MatherT

KamuraT

TakagiY

1999 The elongin B ubiquitin homology domain. Identification of Elongin B sequences important for interaction with Elongin C. J Biol Chem 274 13629 13636

38. HuthoffH

MalimMH

2007 Identification of amino acid residues in APOBEC3G required for regulation by human immunodeficiency virus type 1 Vif and Virion encapsidation. J Virol 81 3807 3815

39. ShiJ

GhirlandoR

BeavilRL

BeavilAJ

KeownMB

1997 Interaction of the low-affinity receptor CD23/Fc epsilonRII lectin domain with the Fc epsilon3-4 fragment of human immunoglobulin E. Biochemistry 36 2112 2122

40. DelaglioF

GrzesiekS

VuisterGW

ZhuG

PfeiferJ

1995 NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6 277 293

41. GoddardTDK

GD

2007 SPARKY 3. University of California, San Francisco

42. JohnsonBA

2004 Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol Biol 278 313 352

43. PlattEJ

WehrlyK

KuhmannSE

ChesebroB

KabatD

1998 Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol 72 2855 2864

44. SimonJH

SoutherlingTE

PetersonJC

MeyerBE

MalimMH

1995 Complementation of vif-defective human immunodeficiency virus type 1 by primate, but not nonprimate, lentivirus vif genes. J Virol 69 4166 4172

45. NewmanEN

HolmesRK

CraigHM

KleinKC

LingappaJR

2005 Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity. Curr Biol 15 166 170

46. ZhengN

SchulmanBA

SongL

MillerJJ

JeffreyPD

2002 Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 416 703 709

47. ThompsonJD

GibsonTJ

HigginsDG

2002 Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics Chapter 2 Unit 2 3

48. KelleyLA

SternbergMJ

2009 Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4 363 371

49. GouetP

RobertX

CourcelleE

2003 ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31 3320 3323

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Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

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