Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest

English version České info

HIV-1 Viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/stress checkpoint. Recently, we and several other groups showed that Vpr performs this activity by recruiting the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. While recruitment of this E3 ubiquitin ligase complex has been shown to be required for G2 arrest, the subcellular compartment where this complex forms and functionally acts is unknown. Herein, using immunofluorescence and confocal microscopy, we show that Vpr forms nuclear foci in several cell types including HeLa cells and primary CD4+ T-lymphocytes. These nuclear foci contain VPRBP and partially overlap with DNA repair foci components such as γ-H2AX, 53BP1 and RPA32. While treatment with the non-specific ATR inhibitor caffeine or depletion of VPRBP by siRNA did not inhibit formation of Vpr nuclear foci, mutations in the C-terminal domain of Vpr and cytoplasmic sequestration of Vpr by overexpression of Gag-Pol resulted in impaired formation of these nuclear structures and defective G2 arrest. Consistently, we observed that G2 arrest-competent sooty mangabey Vpr could form these foci but not its G2 arrest-defective paralog Vpx, suggesting that formation of Vpr nuclear foci represents a critical early event in the induction of G2 arrest. Indeed, we found that Vpr could associate to chromatin via its C-terminal domain and that it could form a complex with VPRBP on chromatin. Finally, analysis of Vpr nuclear foci by time-lapse microscopy showed that they were highly mobile and stable structures. Overall, our results suggest that Vpr recruits the DDB1-CUL4A (VPRBP) E3 ligase to these nuclear foci and uses these mobile structures to target a chromatin-bound cellular substrate for ubiquitination in order to induce DNA damage/replication stress, ultimately leading to ATR activation and G2 cell cycle arrest.

Vyšlo v časopise: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest. PLoS Pathog 6(9): e32767. doi:10.1371/journal.ppat.1001080
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001080

Souhrn

Zdroje

1. MalimMH

EmermanM

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

2. BachandF

YaoXJ

HrimechM

RougeauN

CohenEA

1999 Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor. J Biol Chem 274 9083 9091

3. LuYL

BennettRP

WillsJW

GorelickR

RatnerL

1995 A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles. J Virol 69 6873 6879

4. SeligL

PagesJC

TanchouV

PreveralS

Berlioz-TorrentC

1999 Interaction with the p6 domain of the gag precursor mediates incorporation into virions of Vpr and Vpx proteins from primate lentiviruses. J Virol 73 592 600

5. MorelletN

BouazizS

PetitjeanP

RoquesBP

2003 NMR structure of the HIV-1 regulatory protein VPR. J Mol Biol 327 215 227

6. MorelletN

RoquesBP

BouazizS

2009 Structure-function relationship of Vpr: biological implications. Curr HIV Res 7 184 210

7. Le RouzicE

BenichouS

2005 The Vpr protein from HIV-1: distinct roles along the viral life cycle. Retrovirology 2 11

8. PlanellesV

JowettJB

LiQX

XieY

HahnB

1996 Vpr-induced cell cycle arrest is conserved among primate lentiviruses. J Virol 70 2516 2524

9. StivahtisGL

SoaresMA

VodickaMA

HahnBH

EmermanM

1997 Conservation and host specificity of Vpr-mediated cell cycle arrest suggest a fundamental role in primate lentivirus evolution and biology. J Virol 71 4331 4338

10. ZimmermanES

ShermanMP

BlackettJL

NeidlemanJA

KreisC

2006 Human immunodeficiency virus type 1 Vpr induces DNA replication stress in vitro and in vivo. J Virol 80 10407 10418

11. WardJ

DavisZ

DeHartJ

ZimmermanE

BosqueA

2009 HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response. PLoS Pathog 5 e1000613

12. RichardJ

SindhuS

PhamTN

BelzileJP

CohenEA

2010 HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell-mediated killing. Blood 115 1354 1363

13. RoshalM

KimB

ZhuY

NghiemP

PlanellesV

2003 Activation of the ATR-mediated DNA damage response by the HIV-1 viral protein R. J Biol Chem 278 25879 25886

14. ZimmermanES

ChenJ

AndersenJL

ArdonO

DehartJL

2004 Human immunodeficiency virus type 1 Vpr-mediated G2 arrest requires Rad17 and Hus1 and induces nuclear BRCA1 and gamma-H2AX focus formation. Mol Cell Biol 24 9286 9294

15. CimprichKA

CortezD

2008 ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol 9 616 627

16. McGowanCH

RussellP

2004 The DNA damage response: sensing and signaling. Curr Opin Cell Biol 16 629 633

17. LaiM

ZimmermanES

PlanellesV

ChenJ

2005 Activation of the ATR pathway by human immunodeficiency virus type 1 Vpr involves its direct binding to chromatin in vivo. J Virol 79 15443 15451

18. AndersenJL

ZimmermanES

DeHartJL

MuralaS

ArdonO

2005 ATR and GADD45alpha mediate HIV-1 Vpr-induced apoptosis. Cell Death Differ 12 326 334

19. BelzileJP

DuisitG

RougeauN

MercierJ

FinziA

2007 HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4A(VPRBP) E3 Ubiquitin Ligase. PLoS Pathog 3 e85

20. DeHartJL

ZimmermanES

ArdonO

Monteiro-FilhoCM

ArganarazER

2007 HIV-1 Vpr activates the G2 checkpoint through manipulation of the ubiquitin proteasome system. Virol J 4 57

21. HreckaK

GierszewskaM

SrivastavaS

KozaczkiewiczL

SwansonSK

2007 Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle. Proc Natl Acad Sci U S A 104 11778 11783

22. Le RouzicE

BelaidouniN

EstrabaudE

MorelM

RainJC

2007 HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle 6 182 188

23. SchrofelbauerB

HakataY

LandauNR

2007 HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1. Proc Natl Acad Sci U S A 104 4130 4135

24. TanL

EhrlichE

YuXF

2007 DDB1 and Cul4A are required for human immunodeficiency virus type 1 Vpr-induced G2 arrest. J Virol 81 10822 10830

25. WenX

DuusKM

FriedrichTD

de NoronhaCM

2007 The HIV1 protein Vpr acts to promote G2 cell cycle arrest by engaging a DDB1 and Cullin4A-containing ubiquitin ligase complex using VprBP/DCAF1 as an adaptor. J Biol Chem 282 27046 27057

26. BelzileJP

RichardJ

RougeauN

XiaoY

CohenEA

2010 HIV-1 Vpr induces the K48-linked polyubiquitination and proteasomal degradation of target cellular proteins to activate ATR and promote G2 arrest. J Virol 84 3320 3330

27. SrivastavaS

SwansonSK

ManelN

FlorensL

WashburnMP

2008 Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection. PLoS Pathog 4 e1000059

28. SharovaN

WuY

ZhuX

StranskaR

KaushikR

2008 Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction. PLoS Pathog 4 e1000057

29. BergamaschiA

AyindeD

DavidA

Le RouzicE

MorelM

2009 The human immunodeficiency virus type 2 Vpx protein usurps the CUL4A-DDB1 DCAF1 ubiquitin ligase to overcome a postentry block in macrophage infection. J Virol 83 4854 4860

30. BoltonDL

LenardoMJ

2007 Vpr cytopathicity independent of G2/M cell cycle arrest in human immunodeficiency virus type 1-infected CD4+ T cells. J Virol 81 8878 8890

31. DepienneC

RoquesP

CreminonC

FritschL

CasseronR

2000 Cellular distribution and karyophilic properties of matrix, integrase, and Vpr proteins from the human and simian immunodeficiency viruses. Exp Cell Res 260 387 395

32. YaoXJ

SubbramanianRA

RougeauN

BoisvertF

BergeronD

1995 Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation. J Virol 69 7032 7044

33. Di MarzioP

ChoeS

EbrightM

KnoblauchR

LandauNR

1995 Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J Virol 69 7909 7916

34. LuYL

SpearmanP

RatnerL

1993 Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions. J Virol 67 6542 6550

35. ZhaoLJ

MukherjeeS

NarayanO

1994 Biochemical mechanism of HIV-I Vpr function. Specific interaction with a cellular protein. J Biol Chem 269 15577 15582

36. SubbramanianRA

YaoXJ

DilhuydyH

RougeauN

BergeronD

1998 Human immunodeficiency virus type 1 Vpr localization: nuclear transport of a viral protein modulated by a putative amphipathic helical structure and its relevance to biological activity. J Mol Biol 278 13 30

37. VodickaMA

KoeppDM

SilverPA

EmermanM

1998 HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev 12 175 185

38. JacquotG

Le RouzicE

DavidA

MazzoliniJ

BouchetJ

2007 Localization of HIV-1 Vpr to the nuclear envelope: impact on Vpr functions and virus replication in macrophages. Retrovirology 4 84

39. KamataM

AidaY

2000 Two putative alpha-helical domains of human immunodeficiency virus type 1 Vpr mediate nuclear localization by at least two mechanisms. J Virol 74 7179 7186

40. FritzJV

DidierP

ClammeJP

SchaubE

MuriauxD

2008 Direct Vpr-Vpr interaction in cells monitored by two photon fluorescence correlation spectroscopy and fluorescence lifetime imaging. Retrovirology 5 87

41. MahalingamS

AyyavooV

PatelM

Kieber-EmmonsT

WeinerDB

1997 Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr. J Virol 71 6339 6347

42. MahalingamS

CollmanRG

PatelM

MonkenCE

SrinivasanA

1995 Functional analysis of HIV-1 Vpr: identification of determinants essential for subcellular localization. Virology 212 331 339

43. ShermanMP

de NoronhaCM

HeuschMI

GreeneS

GreeneWC

2001 Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr. J Virol 75 1522 1532

44. de NoronhaCM

ShermanMP

LinHW

CavroisMV

MoirRD

2001 Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr. Science 294 1105 1108

45. SoderbergO

GullbergM

JarviusM

RidderstraleK

LeuchowiusKJ

2006 Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3 995 1000

46. XiaoY

ChenG

RichardJ

RougeauN

LiH

2008 Cell-surface processing of extracellular human immunodeficiency virus type 1 Vpr by proprotein convertases. Virology 372 384 397

47. LaiJS

HerrW

1992 Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. Proc Natl Acad Sci U S A 89 6958 6962

48. MisteliT

2007 Beyond the sequence: cellular organization of genome function. Cell 128 787 800

49. MisteliT

SoutoglouE

2009 The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol 10 243 254

50. MateraAG

Izaguire-SierraM

PraveenK

RajendraTK

2009 Nuclear bodies: random aggregates of sticky proteins or crucibles of macromolecular assembly? Dev Cell 17 639 647

51. ZhangS

PointerD

SingerG

FengY

ParkK

1998 Direct binding to nucleic acids by Vpr of human immunodeficiency virus type 1. Gene 212 157 166

52. JakobB

SplinterJ

DuranteM

Taucher-ScholzG

2009 Live cell microscopy analysis of radiation-induced DNA double-strand break motion. Proc Natl Acad Sci U S A 106 3172 3177

53. FritzJV

DujardinD

GodetJ

DidierP

De MeyJ

2010 HIV-1 Vpr oligomerization but not that of Gag directs the interaction between Vpr and Gag. J Virol 84 1585 1596

54. YaoXJ

MoulandAJ

SubbramanianRA

ForgetJ

RougeauN

1998 Vpr stimulates viral expression and induces cell killing in human immunodeficiency virus type 1-infected dividing Jurkat T cells. J Virol 72 4686 4693

55. HoshinoS

SunB

KonishiM

ShimuraM

SegawaT

2007 Vpr in plasma of HIV type 1-positive patients is correlated with the HIV type 1 RNA titers. AIDS Res Hum Retroviruses 23 391 397

56. BarilM

RacineME

PeninF

LamarreD

2009 MAVS dimer is a crucial signaling component of innate immunity and the target of hepatitis C virus NS3/4A protease. J Virol 83 1299 1311

57. LavalleeC

YaoXJ

LadhaA

GottlingerH

HaseltineWA

1994 Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles. J Virol 68 1926 1934

58. KobingerGP

BorsettiA

NieZ

MercierJ

DanielN

1998 Virion-targeted viral inactivation of human immunodeficiency virus type 1 by using Vpr fusion proteins. J Virol 72 5441 5448

59. DubeM

RoyBB

Guiot-GuillainP

MercierJ

BinetteJ

2009 Suppression of Tetherin-Restricting Activity on HIV-1 Particle Release Correlates with Localization of Vpu in the trans-Golgi Network. J Virol