Skowronski samhd1-20121105

Innate immunity and HIV infection

Jacek Skowronski, PhDCase Western Reserve University

November 5, 2012

1. Background: HIV accessory virulence factors Vpx and Vpr

2. Vpx and Vpr function through CRL4DCAF1 E3 ubiquitin ligase

3. Identification of cellular protein, SAMHD1, Vpx targets for degradation via CRL4DCAF1

4. SAMHD1 inhibits HIV infection in quiescent cells

5. Ongoing SAMHD1 studies

vpr

envpol

gag

vpx, vpr

envpol

gagHIV-1

HIV-2/SIVsm

vif

vif

vpu

nef

nef

The innate immune system protects from viral infection.

Short J A Bioscience Horizons 2009;2:212-224

The innate immune system matrix.

Block ISGfunction

Block IFNsignaling

IFN – Rdecoys

Block IFNsynthesis



TRIM5TRIM28/KAP1APOBEC3G/FTetherin/BST2Schlafen 11XYZ??



HIV:EvadesCounteractsIgnores


TRIM5TRIM28/KAP1APOBEC3G/FTetherin/BST2Schlafen 11X ??

vpr

envpol

gag

vpx, vpr

envpol

gagHIV-1

HIV-2/SIVsm

vif

vif

vpu

nef

nef

HIV accessory proteins program degradation of restriction factors

Target:

APOBEC3G/FTetherinXYZ??

Accessory protein

VifVpuVpr, Vpx

E3 Ubiqitin ligase:

CRL5SCF--TrCPCRL4-DCAF1

–> degradation by proteasome


E3

Function and regulation of E3 Ubiquitin ligases

target

targetUb Ub

targetUb UbUbUb

26 SproteasomeE2

Ub

E2 E1

Ub

E1

E3

Nedd8

Nedd8 CSN

Roc1 (CyP F)

vpr

envpol

gag

vpx, vpr

envpol

gagHIV-1

HIV-2/SIVsm

Target:

APOBEC3G/FTetherinXYZ??

Accessory protein

VifVpuVpr, Vpx

E3 Ubiqitin ligase:

CRL5SCF--TrCPCRL4-DCAF1


–> degradation by proteasome

NMR

X-RAY

WAGVEAIIR-----ILQQLPFIHFRIGCRHSRIGV-TRQRRARNG--ASRS*LEGAGELIR-----ILQRALFMHFRGGCIHSRIGQ-PGGGNPLSAIPPSRSML*QGMSPSYVKYRYLCLIQKALFMHCKKGCRCLGEGHGAGGWRPGPPPPPPPGLA*

MEQAP-EDQGPQ--REPYNEWTLELLEELKSEAVRHFPRIWLHNLGRHIYETYGDTMEERPPENEGPQ--REPWDEWVVEVLEELKEEALKHFDPRLLTALGNHIYNRHGDT

MSDPRERIPPGNSGEETIGEAF-EWLNRTVEEINREAVNHLPRELIFQVWQRSWEYWHDE

HIV-1 VprSIVmac VprSIVmac Vpx

Vpr (HIV-1)

Vpr (SIVmac)

Vpx (SIVmac)

Vpr and Vpx functions:

DNA damage checkpoint (G2)

+++

+++

–

+

+++++

–

Transduction of Macrophages/DC

Transcription

++

?

?

Vpx overcomes inhibition of HIV infection of MDM and MDDC

Negre et al. (2000) Gene Ther 7:1613Goujon et al. (2007) Retrovirology 4:2Kaushik et al. (2009) Cell Host Microbe 6:68Pertel et al. (2011) Retrovirology 8:49

Substrate

specificity

DC

AF1

DDB1DDA1

Vpr/Vpx

CRL4 E3 architecture

Vpx/Vpr-bound protein complex

Substrate

specificity

Cat

alyt

ic c

ore DDB1

Cul

lin 4

CSNDCAF1

Vpx, VprEpitope tagging

Expression

Affinity purification

LC-MS/MS(shotgun/MudPIT)

Vpx/Vpr Complex

Identification

Vpx and Vpr target CRL4 DCAF1 E3 Ubiquitin ligase

Substrate

specificity

Cat

alyt

ic c

ore

“WD40”

DDB1

Cul

lin 4

“WD40” substrate receptors:DCAF1-19DDB2CSACdt2VprBP/DCAF1

Architecture of Cullin 4-based E3 ubiquitin ligase complexes

Vpr/Vpx bound complex:

Substrate

specificity

DC

AF1

DDB1DDA1

Vpr/Vpx

VprBP

10% sucrose 40%

232 660 kD66

DCAF1

DDB1DDA1

DCAF1

DDB1DDA1Vpr

Blot:

IP: VprBP

IP: Vpr

CSN

Vpr (&Vpx) stimulates catalysis by Cul4DCAF1 E3

DDB1DCAF1

DCAF1DDB1

Cul

lin 4

Cul

lin 4

E3 active

or inactive ?Vpr/Vpx

DCAF1DDB1

Cul

lin 4


DDB1C

ullin

4

tag

DCAF1 DCAF1DDB1

Cul

lin 4

tagVpr

“WD40”: DCAF1

26 Sproteasome

target

UbUb

UbUb

target

target

Ub

E2

E2Ub

E3E3

Nedd8

Nedd8

Assembly of Cul4DCAF1 E3 + / - Vpr in HEK 293T cells

Purification via DCAF1 subunit

Incubation with E2 & Ubiquitin

SDS PAGE for poly-Ub Cul4

CSN

Roc1 (CyP F)


DDB1C

ullin

4

tag

DCAF1 DCAF1DDB1

Cul

lin 4

tagVpr

“WD40”: DCAF1

Vpr:E2:

– – + – +– – – + +

Cul4

Cul4(Ub)n

1 2 3 4 5

26 Sproteasome

target

UbUb

UbUb

target

target

Ub

E2

E2Ub

E3E3

Nedd8

Nedd8

f–Vpr:f–VprBP:f–DDB2:m-Cul4A:Vpr:VprBP:

+–––+–

+––++–

+––+++

–––+++

–+–––+

–+–+–+

–+–+++

–––++–

–––+++

––++++

––+++–

––++–+

––+–––

Cul4

Cul4

Nedd8

Ext

ract

IP

IP: f-Vpr f-DCAF1 f-DDB2

*

*

*

Vpr (&Vpx) elevates neddylation of DCAF1 associated Cul4

DDB1

Cul

lin 4

tag

DDB2DDB1

Cul

lin 4

tag

VprBP VprBPDDB1

Cul

lin 4

tagVpr

“WD40”: VprBP

DDB1

Cul

lin 4

tag

DDB2DDB1

Cul

lin 4

tag

DCAF1 DCAF1DDB1

Cul

lin 4

tagVpr

“WD40”: DCAF1 “WD40”: DDB2

E3E3

Nedd8

Nedd8

E3E3

Nedd8

Nedd8 CSN

Roc1 (CyP F)

VprVpx

Vpx/Vpr usurp CRL4DCAF1 to destroy novel restriction factors

E3

Ub UbUb UbUbUb

26 SproteasomeE2

Ub

E2 E1

Ub

E1

E3

Nedd8

Nedd8 CSN

Roc1 (CyP F)

VpxVpr

Novel Restriction Factors for HIV/SIV

Vpx/Vpr usurp CRL4DCAF1 to destroy novel restriction factors

Le Rouzic et al. 2007 Cell Cycle 6:182-8Schröfelbauer et al. 2007. PNAS 104::4130-5Hrecka et al. 2007. PNAS 104:11778-83De Hart et al. 2007 Virol J. 4:57Wen et al. 2007 JBC 282:27046-57Srivastava et al. 2009 PLoS Path. 4:e1000059…

1. Substrate trapping by Vpx/Vpr on CRL4DCAF1

1. Global analysis of DC proteomes following SIV VLP(Vpx)infection

Searches for substrates recruited by Vpr/Vpx to CRL4DCAF1 E3

Substrate trapping on CRL4DCAF1 by Vpx/Vpr

transient co-expression

MudPIT

affinity purification of E3 complexes

FLAG-HA-

Vpx

FLAG-HA-

Vpr (H/S)

DDB1C

ullin

4

DDA1

FLAG-HA-

CRL4DCAF1–Vpx

DDB1

Cul

lin4

DDA1

FLAG-HA-

CRL4DCAF1–Vpr

DDB1

Cul

lin4

DDA1

FLAG- HA-

CRL4DCAF1

Cellular proteins loaded by Vpx onto CRL4DCAF1 by Vpx

Putative Vpx substrates trapped on CRL4DCAF1

CD14+ monocytes

GMCSF, IL-4

MDDC

(+/- type I IFN)

SIV VLP +/- Vpx

Extracts (cyt or nuc)

MudPIT

MudPIT analysis of MDDC proteomes following SIV VLP(Vpx) infection

MDDC extract

SIV VLP IFN SAMHD1 A3C A3A spectra proteins

Cytoplasmic – – 35 0 0 14085 1048

Nuclear – – 17 10 0 18077 1992

Cytoplasmic Vpx – 0 2 0 25878 1536

Nuclear Vpx – 0 5 0 15803 1714

Cytoplasmic – IFN 65 1 7 28047 1912

Nuclear – IFN 39 6 2 16962 2018

Cytoplasmic Vpx IFN 0 0 13 33280 2158

Nuclear Vpx IFN 0 10 2 25619 2498

Analysis of MDDC proteomes identifies SAMHD1 as Vpx target

MDDC extract

SIV VLP IFN SAMHD1 A3C A3A spectra proteins

Cytoplasmic – – 35 0 0 14085 1048

Nuclear – – 17 10 0 18077 1992

Cytoplasmic Vpx – 0 2 0 25878 1536

Nuclear Vpx – 0 5 0 15803 1714

Cytoplasmic – IFN 65 1 7 28047 1912

Nuclear – IFN 39 6 2 16962 2018

Cytoplasmic Vpx IFN 0 0 13 33280 2158

Nuclear Vpx IFN 0 10 2 25619 2498

Analysis of MDDC proteomes also identifies SAMHD1 as Vpx target

SAMHD1 inhibits HIV infection and

is targeted by Vpx for degradation via CRL4DCAF1

Vpx-recruits SAMHD1 to CRL4DCAF1 complex

Vpx programs SAMHD1 degradation via CRL4DCAF1 in MDM

SAMHD1 inhibits HIV/SIV infection in macrophages

MDM

D0 Monocytes +GMCSFD3 VLPVpxD6 siRNAD9 infect/westernD10 Q-PCRD12 FACS

Protocol

D0 Monocytes +GMCSFD3 VLPVpxD6 siRNAD9 infect/westernD10 Q-PCRD12 FACS



Conclusions

1. SAMHD1 inhibits HIV/SIV infection in macrophages, and other monocyte derived cell lineages.

2. SAMHD1 inhibits infection by interfering with viral cDNA synthesis.

3. SAMHD1 restriction is counteracted by Vpx.

4. Vpx targets SAMHD1 for proteasomal degradation via CRL4DCAF1 E3 Ub ligase.

Hrecka et al. 2011 Nature 474:658-61Laguette et al. 2011 Nature 474:654-7Berger et al. 2011 PLoS Pathogens 7:e1002425

IFN

Viral cDNA

RT

SAMHD1 (626aa)

dN + PPPdNTP

endo-immuno-stimulatory DNA

?

SAM HD

COG1078allosite

anti-inflamatory(AGS)

anti-HIV

SAMHD11. dGTP-dependent dNTPase2. Located in nucleus3. Expressed in most leukocytes (HSC, B,

T, monocytes)

Anti-inflamatory5. Loss of function (lof) SAMHD1 mutations

are associated with autoimmune inflammatory condition (AGS) resembling congenital viral infection.

6. AGS is also caused by lof mutations in nucleases TREX1 and RNAseH2.

7. SAMHD1, TREX1 and RNAseH2 prevent accumulation of cellular immuno-stimulatory DNA that activates type I IFN response via cytoplasmic sensors.

Anti-viral7. Inhibits HIV/SIV cDNA synthesis by

suppressing dNTP pools below the threshold required for RT to function efficiently.

SAMHD1: short story

Outstanding questions:

Anti-viral:• SAMHD1 functions important for anti–viral function, besides catalytic activity.

• is SAMHD1 proximity to RTC important • RTC recognition• is restriction regulated• how does Vpx counteract SAMHD1

• SAMHD1 and innate response to HIV• does SAMHD1 prevent HIV triggering of innate sensors and type I IFN response

in monocytes/DC/macrophages?• Identity of cytoplasmic sensors that mediate this response

Anti-inflamatory• what is the nature of immunostimulatory DNA whose accumulation SAMHD1 prevents?• cytoplasmic sensors that mediate type I interferon response

Collaborators:

Case:David McDonaldMarcin GolczakMichael Lederman

Stowers Institute:Mike WashburnLaurence FlorensSelene K . Swanson

U. Pitt & PCHPI:Angela GronenbornJoanne YehJinwoo Ahn

Skirball Inst. & HHMI:Dan LittmanNicolas Manel

Case:

Caili HaoKasia HreckaSarabpreet KaurSheeba RasheediChuanping WangJunpeng Yan

CSHL

Magda Gierszewska*Smita Srivastava*

Documents

Skowronski samhd1-20121105