Third Annual Mitchell Memorial Lecture hosted by the UC San Diego AIDS Research Institute. Jacek Skowronski, PhD, of Case Western Reserve University

1. Innate immunity and HIV infection
Jacek Skowronski, PhD
Case Western Reserve University
November 5, 2012

2. vif vpu
HIV-1 gag
pol
env
vpr nef
vif
gag
HIV-2/SIVsm pol
env
vpx, vpr nef
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

3. The innate immune system protects from viral infection.
Short J A Bioscience Horizons 2009;2:212-224

4. The innate immune system matrix.
Block IFN
synthesis
IFN – R
decoys
Block IFN
signaling
Block ISG
function
Short J A Bioscience Horizons 2009;2:212-224

5. The innate immune system matrix.
TRIM5
TRIM28/KAP1
APOBEC3G/F
Tetherin/BST2
Schlafen 11
XYZ??
Short J A Bioscience Horizons 2009;2:212-224

6. The innate immune system matrix.
TRIM5 HIV:
TRIM28/KAP1 Evades
APOBEC3G/F Counteracts
Tetherin/BST2 Ignores
Schlafen 11
X ??
Short J A Bioscience Horizons 2009;2:212-224

7. HIV accessory proteins program degradation of restriction factors
vif vpu
HIV-1 gag
pol
env
vpr nef
vif
gag
HIV-2/SIVsm pol
env
vpx, vpr nef
Target: Accessory protein E3 Ubiqitin ligase:
APOBEC3G/F Vif CRL5
Tetherin Vpu SCF--TrCP
XYZ?? Vpr, Vpx CRL4-DCAF1
–> degradation by proteasome

8. HIV accessory proteins program degradation of restriction factors
Function and regulation of E3 Ubiquitin ligases
Roc1 target
Nedd8 (CyP F)
Ub
E2 E1 26 S
proteasome
Ub
E3 E3
E2 E1
Ub UbUbUbUbUb
Nedd8 CSN
target target

9. HIV accessory proteins program degradation of restriction factors
HIV-1 gag
pol
env
vpr
gag
HIV-2/SIVsm pol
env
vpx, vpr
Target: Accessory protein E3 Ubiqitin ligase:
APOBEC3G/F Vif CRL5
Tetherin Vpu SCF--TrCP
XYZ?? Vpr, Vpx CRL4-DCAF1
–> degradation by proteasome

10. Vpr and Vpx functions:
DNA damage Transduction
Transcription
checkpoint (G2) of Macrophages/DC
Vpr (HIV-1) +++ ++ +
Vpr (SIVmac) +++ ? –
Vpx (SIVmac) – ? +++++
HIV-1 Vpr MEQAP-EDQGPQ--REPYNEWTLELLEELKSEAVRHFPRIWLHNLGRHIYETYGDTWAGVEAIIR-----ILQQLPFIHFRIGCRHSRIGV-TRQRRARNG--ASRS*
SIVmac Vpr MEERPPENEGPQ--REPWDEWVVEVLEELKEEALKHFDPRLLTALGNHIYNRHGDTLEGAGELIR-----ILQRALFMHFRGGCIHSRIGQ-PGGGNPLSAIPPSRSML*
SIVmac Vpx MSDPRERIPPGNSGEETIGEAF-EWLNRTVEEINREAVNHLPRELIFQVWQRSWEYWHDEQGMSPSYVKYRYLCLIQKALFMHCKKGCRCLGEGHGAGGWRPGPPPPPPPGLA*
  
X-RAY NMR




11. Vpx overcomes inhibition of HIV infection of MDM and MDDC
Negre et al. (2000) Gene Ther 7:1613
Goujon et al. (2007) Retrovirology 4:2
Kaushik et al. (2009) Cell Host Microbe 6:68
Pertel et al. (2011) Retrovirology 8:49

12. Vpx and Vpr target CRL4 DCAF1 E3 Ubiquitin ligase
CRL4 E3
Vpx/Vpr
architecture
Complex
Identification
DDB1
Catalytic core
Cullin 4
specificity
Substrate
CSN
DCAF1
Vpx, Vpr
Epitope tagging
Expression Vpx/Vpr-bound
protein complex
Affinity purification DDA1
DDB1
DCAF1
specificity
Substrate
LC-MS/MS
Vpr/
(shotgun/MudPIT)
Vpx

13. Architecture of Cullin 4-based E3 ubiquitin ligase complexes
“WD40” substrate receptors:
Catalytic core DDB1 DCAF1-19
Cullin 4
specificity
Substrate
DDB2
CSN
CSA
“WD40”
Cdt2
VprBP/DCAF1
10% sucrose 40%
Vpr/Vpx bound complex:
Blot: 66 232 660 kD
DDA1 DCAF1
DDB1 IP: DDB1
DCAF1
VprBP
specificity
Substrate
DDA1 VprBP
Vpr/ DCAF1
Vpx
IP: DDB1
Vpr DDA1
Vpr

14. Vpr (&Vpx) stimulates catalysis by Cul4DCAF1 E3
E3
active
or inactive ?
DDB1
Vpr/Vpx
DCAF1
DDB1 DDB1
Cullin 4
Cullin 4
DCAF1 DCAF1
Cullin 4

15. Vpr (&Vpx) stimulates catalysis by Cul4DCAF1 E3
Roc1 target Assembly of Cul4DCAF1 E3
Nedd8 (CyP F)
+ / - Vpr in HEK 293T cells
E2
Ub
E3 E3 Purification via DCAF1 subunit
E2
CSN Incubation with E2 & Ubiquitin
Nedd8
Ub
target SDS PAGE for poly-Ub Cul4
26 S
DDB1 DDB1
proteasome
Cullin 4
Cullin 4
Ub DCAF1
DCAF1
Ub
Ub
Vpr
Ub tag tag
target
“WD40”: DCAF1

16. Vpr (&Vpx) stimulates catalysis by Cul4DCAF1 E3
Vpr: – – + – +
target E2: – – – + +
Nedd8
E2
Ub
E3 E3
Cul4(Ub)n
E2
Cul4
Nedd8
Ub
target
1 2 3 4 5
26 S
DDB1 DDB1
Cullin 4
proteasome
Cullin 4
Ub DCAF1
DCAF1
Ub
Ub
Vpr
Ub tag tag
target
“WD40”: DCAF1

17. Vpr (&Vpx) elevates neddylation of DCAF1 associated Cul4
IP: f-Vpr f-DCAF1 f-DDB2
Nedd8 f–Vpr: + + + – – – – – – – – – –
f–VprBP: – – – – + + + – – – – – –
f–DDB2: – – – – – – – – + + + + –
m-Cul4A: – + + + – + + + – + + + +
Vpr: + + + + – – + + – + – + +
E3 E3 VprBP: – – + + + + + – – – + + +
Extract
Cul4 *
Nedd8 Cul4 *
IP
Nedd8
*
DDB1 DDB1 DDB1
Cullin 4
Cullin 4
Cullin 4
DCAF1
VprBP DCAF1
VprBP DDB2
tag
Vpr
tag tag
“WD40”: VprBP
DCAF1 “WD40”: DDB2

18. Vpx/Vpr usurp CRL4DCAF1 to destroy novel restriction factors
Roc1
Nedd8 (CyP F)
Vpr
E3 Vpx E3
Nedd8 CSN

19. Vpx/Vpr usurp CRL4DCAF1 to destroy novel restriction factors
Novel Restriction
Factors for HIV/SIV
Roc1
Nedd8 (CyP F)
Ub
E1 26 S
Vpx E2 proteasome
Ub
E3 Vpr E3
E2 E1
Ub UbUbUbUbUb
Nedd8 CSN
Le Rouzic et al. 2007 Cell Cycle 6:182-8
Schröfelbauer et al. 2007. PNAS 104::4130-5
Hrecka et al. 2007. PNAS 104:11778-83
De Hart et al. 2007 Virol J. 4:57
Wen et al. 2007 JBC 282:27046-57
Srivastava et al. 2009 PLoS Path. 4:e1000059
…

20. Searches for substrates recruited by Vpr/Vpx to CRL4DCAF1 E3
1. Substrate trapping by Vpx/Vpr on CRL4DCAF1
1. Global analysis of DC proteomes following SIV VLP(Vpx)
infection

21. Substrate trapping on CRL4DCAF1 by Vpx/Vpr
Vpx CRL4DCAF1–Vpx CRL4DCAF1 CRL4DCAF1–Vpr Vpr (H/S)
DDA1 DDA1 DDA1
DDB1 DDB1 DDB1
Cullin 4
Cullin 4
Cullin 4
HA- HA-
HA- FLAG- HA- FLAG-
FLAG- FLAG- FLAG-
HA-
transient co-expression
affinity purification of E3 complexes
MudPIT

22. Cellular proteins loaded by Vpx onto CRL4DCAF1 by Vpx

23. Putative Vpx substrates trapped on CRL4DCAF1

24. MudPIT analysis of MDDC proteomes following
SIV VLP(Vpx) infection
CD14+ monocytes
GMCSF, IL-4
MDDC
(+/- type I IFN)
SIV VLP +/- Vpx
Extracts
(cyt or nuc)
MudPIT

25. Analysis of MDDC proteomes identifies SAMHD1 as Vpx
target
MDDC SIV VLP IFN SAMHD1 A3C A3A  spectra  proteins
extract
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

26. Analysis of MDDC proteomes also identifies SAMHD1 as Vpx target
MDDC SIV VLP IFN SAMHD1 A3C A3A  spectra  proteins
extract
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

27. SAMHD1 inhibits HIV infection
and
is targeted by Vpx for degradation via CRL4DCAF1

28. Vpx-recruits SAMHD1 to CRL4DCAF1 complex

29. Vpx programs SAMHD1 degradation via CRL4DCAF1 in MDM

30. SAMHD1 inhibits HIV/SIV infection in macrophages
MDM
D0 Monocytes +GMCSF
D3 VLPVpx
D6 siRNA
D9 infect/western
D10 Q-PCR
D12 FACS

31. SAMHD1 inhibits HIV/SIV infection in macrophages
Protocol
D0 Monocytes +GMCSF
D3 VLPVpx
D6 siRNA
D9 infect/western
D10 Q-PCR
D12 FACS

32. SAMHD1 inhibits HIV/SIV infection in macrophages

33. 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 474:658-61
Laguette et al. 2011 Nature 474:654-7
Berger et al. 2011 PLoS Pathogens 7:e1002425

34. SAMHD1: short story
SAMHD1 SAMHD1 (626aa)
1. dGTP-dependent dNTPase
HD
2. Located in nucleus SAM
3. Expressed in most leukocytes (HSC, B,
allo COG1078
T, monocytes) site
Anti-inflamatory
5. Loss of function (lof) SAMHD1 mutations dNTP dN + PPP
are associated with autoimmune
inflammatory condition (AGS) resembling
congenital viral infection.
6. AGS is also caused by lof mutations in
?
nucleases TREX1 and RNAseH2. RT
7. SAMHD1, TREX1 and RNAseH2 prevent
accumulation of cellular immuno-
stimulatory DNA that activates type I IFN endo-immuno-
stimulatory DNA Viral cDNA
response via cytoplasmic sensors.
Anti-viral IFN
7. Inhibits HIV/SIV cDNA synthesis by
suppressing dNTP pools below the
threshold required for RT to function anti-inflamatory anti-HIV
efficiently. (AGS)

35. 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

36. Collaborators:
Case: Case:
David McDonald
Caili Hao Marcin Golczak
Michael Lederman
Kasia Hrecka
Sarabpreet Kaur Stowers Institute:
Sheeba Rasheedi Mike Washburn
Chuanping Wang Laurence Florens
Junpeng Yan Selene K . Swanson
U. Pitt & PCHPI:
CSHL Angela Gronenborn
Joanne Yeh
Jinwoo Ahn
Magda Gierszewska*
Smita Srivastava* Skirball Inst. & HHMI:
Dan Littman
Nicolas Manel