Impaired dNTPase Activity of SAMHD1 by Phosphomimetic Mutation ...
Skowronski samhd1-20121105
description
Transcript of 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
Short J A Bioscience Horizons 2009;2:212-224
The innate immune system matrix.
TRIM5TRIM28/KAP1APOBEC3G/FTetherin/BST2Schlafen 11XYZ??
Short J A Bioscience Horizons 2009;2:212-224
The innate immune system matrix.
HIV:EvadesCounteractsIgnores
Short J A Bioscience Horizons 2009;2:212-224
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
HIV accessory proteins program degradation of restriction factors
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
HIV accessory proteins program degradation of restriction factors
–> 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
Vpr (&Vpx) stimulates catalysis by Cul4DCAF1 E3
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)
Vpr (&Vpx) stimulates catalysis by Cul4DCAF1 E3
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
SAMHD1 inhibits HIV/SIV infection in macrophages
SAMHD1 inhibits HIV/SIV infection in macrophages
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*