HIVs interaction with APOBEC3C (a work in progress) Carsten Mnk - - PowerPoint PPT Presentation

Carsten Münk

Clinic for Gastroenterology, Hepatology and Infectiology Heinrich-Heine-University Düsseldorf

HIV’s interaction with APOBEC3C

(a work in progress)

HIV-1 Vif protein induces degradation of APOBEC3G

infectious HIV-1 non- infectious HIV-1

Münk et al. 2012

Human APOBEC3 proteins

Vif

Structure of Z1c of A3G

hydrophobic five –stranded β sheets (β1-β5) surrounded by six α helices (α1-α6)

zinc atom ssDNA

Jaguva Vasudevan et. al 2013

A3 Vif A3

degradation

Vif binding to APOBEC3s (A3) required for A3 proteasomal degradation

Aydin et al. 2014

Mutations !

Cytidin-Deamination of ssDNA

Encapsidated APOBEC3G deaminates deoxy-cytidines in single stranded viral DNA

Münk et al. 2012

A3G binds the HIV RNA genome, enzymatic activity on ssDNA

Chaurasiya et al. 2013

HIV-1 Δvif HIV-1 wt each line represents an individual PCR on viral DNA

Encapsidated APOBEC3G deaminates deoxy-cytidines in single stranded viral DNA

PCR, 10 h post infection

DÖRRSCHUCK ET AL. 2011

Trinucleotide preferences

Nucleotide preferences of human A3s in virus infection

CCC TCC ACC TTC ATC TTC ATC

A3G binds ssDNA mainly as a dimer

RNA interaction  High Molecular Mass complexes of A3G

Jaguva Vasudevan et. al 2013

Chaurasiya et al. 2013

A3G monomers/dimers convert to oligomers that slowly dissociate

Chelico et al. 2006

APOBEC3G: 3′→5′ ‘slide and jump’ mechanism, no random ‘on and off’ mechanism

HIV FIV

(or Bovine immunodeficiency virus BIV in cattle, Equine infectious anemia virus EIAV in horses/donkeys, Visna in sheep)

APOBEC3s strongly restrict cross-species lentiviral transmissions between distantly related mammals

The balance of APOBEC3 –Vif modulates the viral mutationrate

Münk et al. Viruses 2012

Model of Vif/A3 complexes hypothetical role of oligomerization

Vif Vif A3 A3 degradation + E3 Ligase

active domain

APOBEC3C is Z2 protein

A3C‘s interaction with HIV-1

• 1. where does Vif bind in A3C?
• 2. where does A3C bind in Vif
• 3. antiviral activity of A3C

Structure guide mutagenesis identified Leu72, Phe75, Cys76, Ile79, Leu80, Ser81, Tyr86, Glu106, Phe107 and His111 involved in forming the Vif-interaction interface.

αTubulin Vif-V5 C1 C4 C5 C30

HIV-1 Vif

αTubulin hu A3C CPZ A3C rhe A3C AGM A3C SmmA3C

FeA3Z2b Vif-V5 αTubulin

Species-specific Vif A3C/A3Z2 interactions

huA3C feA3Z2b huA3C feA3Z2b huA3C feA3Z2b HIV-1 Vif

HIV-1 Vif induced degradation:

+ +

• Chimeric A3C/Z2 (feline/human): 46% ident. AA

Vif binding pocket Kitamura et al. 2012

human : mangabey A3C: 76% ident. AA

Human A3C

• Mangab. A3C

Human A3C

• Mangab. A3C

Human A3C

• Mangab. A3C

HuA3C SmmA3C C5 C1 C6 C2 C4 C3 C7 C8 C9 C10

Anti-HA tubulin Anti-v5

HIV1 VIF

• + - + - + - +
• ver exp. 30sec

normal exp. 5sec

HIV-1 Vif induced degradation:

+ +

• Vif binding pocket

Kitamura et al. 2012

Transfection human A3C , rhesus A3C and their chimers with HIV1 VIF

Anti-HA tubulin Anti-v5

HIV1 VIF - + - + - + - + - + - +

• + - +

HuA3C RhA3C RhehuA3C9 RhehuA3C11 RhehuA3C13 RhehuA3C15 RhehuA3C17 RhehuA3C31

In the six chimers, only RheA3C9 and RheA3C11 can be degradated by HIV1 VIF So we can believe the C-terminal of HuA3C is important for HIV1 VIF binding.

85% ident. AA

Human A3C Rhesus A3C Human A3C Rhesus A3C Human A3C Rhesus A3C 1 3 4 5 6 HuA3C RhA3C RhehuA3C9 RhehuA3C11 RhehuA3C13 RhehuA3C15 RhehuA3C17 RhehuA3C31 1 3 4 5 6

HIV-1 Vif induced degradation:

+ + +

• Vif binding pocket

Kitamura et al. 2012

85% ident. AA

HIV-1 Vif binding region in A3C: extended view

New extended Vif binding surface Vif binding surface (Kitamara et al.)

Looks like residues both are forming a cavity where vif binds! We will mutate these co-operative residues to know exactly.

resides between α2 and α3; shown in wheat, color red (> 50% resistance) raspberry (40–45%) indicates the resistance levels of Vif binding to A3C when they are mutated.

APOBEC3-binding sites in HIV-1 Vif

Aydin et al. 2014

VIF B-NL4-3

A3F WX2DRMR W DRMR A3G WXSLVK W SLVK A3G YRHHY F H A3H FX8H YRHHY V IPL L A3F/A3G VXIPLX4L Y L A3F/A3G YXXL TGER W A3F TGERXW LG G I W A3F/A3G LGXGX2IXW TQ AD I A3F/A3G TQX5ADX2I H C Cul5-E3(HCCH) C H S LQYLA BC BOX/CuL5-E3 SLQYLA PPLP L Cul5-E3 (PPLPX4L) EDRW A3F (EDRW)

HIV-1 Vif binding domains

APOBEC3 binding E3 ligase binding N-Term C-Term

1 94 95 193

VIF B-NL4-3

A3F WX2DRMR W DRMR A3G WXSLVK W SLVK A3G YRHHY F H A3H FX8H YRHHY V IPL L A3F/A3G VXIPLX4L Y L A3F/A3G YXXL TGER W A3F TGERXW LG G I W A3F/A3G LGXGX2IXW TQ AD I A3F/A3G TQX5ADX2I H C Cul5-E3(HCCH) C H S LQYLA BC BOX/CuL5-E3 SLQYLA PPLP L Cul5-E3 (PPLPX4L) EDRW A3F (EDRW)

HIV-1 Vif binding domains

APOBEC3 binding E3 ligase binding N-Term C-Term

1 94 95 193

R R A3C ? E D A3C ?

Vif-V5 A3C αTubulin

Determinants in HIV-1 and HIV-2 Vif important for A3C degradation

red color: RR and ED residues in HIV-1 Vif

HIV-1 Vif structure

Vif HIV-1 Plasmids in the lab

A-R37 A-008 B-NL4-3 B-LAI B-026 B-R01 B-102# B-JRCSF C-564 C-748 C-MJ4 D-114 D-158 E-402 F-029 F-019 F-020 G-003 N-CM.02.DJO0131 N-CM.04.CM113 (N-113) N- CM.99.YBF116 (N-116) O-BCF119 (O-119) O-FR.92.VAU (O-VAU) O-RBF127 (O-127) P-L2005 stP (P-PL05)

subtype B Group M

B R01 B NL4-3 D 158 E 402 F 029 F 019

A3C A3G

A3G Vif

• -
• GAPDH

Vif:

B R01 B NL4-3 D 158 E 402 F 029 F 019

A3C

A3C degradation: some Vifs are different

A3C degradation: B-NL4-3: + D-158: ++ F-029:

• B-NL4-3

D-158 F-029 B-NL4-3 D-158 F-029

A3F WX2DRMR W DRMR A3G WXSLVK W SLVK A3G YRHHY F H A3H FX8H YRHHY V IPL L A3F/A3G VXIPLX4L Y L A3F/A3G YXXL TGER W A3F TGERXW LG G I W A3F/A3G LGXGX2IXW TQ AD I A3F/A3G TQX5ADX2I H C Cul5-E3(HCCH) C H S LQYLA BC BOX/CuL5-E3 SLQYLA PPLP L Cul5-E3 (PPLPX4L) EDRW A3F (EDRW)

R R A3C E D A3C  more complex than predicted

193 1

1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5

A3C A3G A3C A3G SIV only HIV only + Vif ∆ Vif

HIV-1 ∆vif luc SIVagm ∆vif luc rel luciferase activity [cps]

+ - + - virions cells + - + - Vif

APOBEC3 α-Tub APOBEC3 α-CA HIV-1 ∆vif luc SIVagm ∆vif luc

A3C A3G A3C A3G

A3C is not antiviral against HIV-1 NL4-3, but strongly inhibits SIVagm Δ Vif

SIV HIV-1

Why SIV, not HIV-1? A3C A3G

Sub-viral localisation of A3C?

SIV HIV1 A3C Vpr.A3C

Zn2+

COOH NH3 COOH NH3

Gly4-Ser

Zn2+

A3C Vpr.A3C

Hypothese I Will core localizing HIV-1 Vpr domain increase the anti HIV-activity of A3C?

Vpr.3C inhibiert HIV Vif-unabhängig Vpr-A3C: more potent and Vif-resistant

Vpr-A3C + Vif Vpr-A3C Vpr-A3C Vpr-A3C + Vif

µg plasmd DNA

• rel. luciferase activity (cps)

viral glycoprotein (VSV-G) viral core fraction: IN, RT, capsid (p24)

Purification of HIV-1 Cores

• ptiprep

Both A3C and Vpr.A3C localize to cores

α-VSV-G α-HA

1 2 3 4 5 6 7 8 9 10 11 Fraktion # % OptiPrep

HIV ∆vif + A3C

60 20

HIV ∆vif + A3C

2000 4000 6000 8000 1 2 3 4 5 6 7 8 9 10 11 1 1,1 1,2 1,3 1,4 RT Aktivität Dichte RT Aktivität [pg/mL] Dichte ρ [g/mL] Fraktion # 1 2 3 4 5 6 7 8 9 10 11

α-VSV-G α-HA

Fraktion #

HIV ∆vif + Vpr.3C

% OptiPrep 60 20

HIV ∆vif + Vpr.3C

2000 4000 6000 8000 10000 1 2 3 4 5 6 7 8 9 10 11 1 1,1 1,2 1,3 1,4 RT Aktivität Dichte RT Aktivität [pg/mL] Dichte ρ [g/mL] Fraktion #

Density Density Fraction Fraction RT activity (pg/ml) RT activity (pg/ml)

Density Density Fraction # Fraction #

α-Tubulin α-HA huA3G huA3C CPZA3C

10ng 150ng pcDNA3.1 10ng 150ng pcDNA3.1 10ng 150ng pcDNA3.1

rhA3C AGM A3C

10ng 150ng pcDNA3.1 10ng 150ng pcDNA3.1

SmmA3C

10ng 150ng pcDNA3.1

FcaA3Z2b

10ng 150ng pcDNA3.1

HIV-1 Luc (3 plasmid system)

A3C determinants required for anti-HIV-1 activity

HuA3C SmmA3C C5 C1 C6 C2 C4 C3 C7 C8 C9 C10

human : mangabey A3C: 76% ident. AA

Human A3C

• Mangab. A3C

Human A3C

• Mangab. A3C

Human A3C

• Mangab. A3C

HOS

PCR (Taq Pol) Sequencing

GA mutations?

SIVagm Δvif +/- APOBEC3

Isolation of total DNA

A3C and SIVagm: no induction of G-to-A mutations

(DNaseI treated)

Is the Zn2+-finger the active domain?

His-66 Cys-97 Glu-68 Cys-100

A3C (Z2)

Zn2+

?

A3G-C term (Z1)

His-257 Cys-288 Cys-291 Glu-256 Zn2+ active site 1 active site 2 HxE------PCxxC HxE------PCxxC

A3G

incorporation in the virion catalytic active site

NH3

A3Cwt: H-X-E-X27-P-C-X2-C H66R: R-X-E-X27-P-C-X2-C E68Q: H-X-Q-X27-P-C-X2-C C98S: H-X-E-X27-P-S-X2-C C101S: H-X-E-X27-P-C-X2-S CC-SS: H-X-E-X27-P-S-X2-S

active site Zn2+

A3C

COOH NH3 COOH

• + - + - + - + - + - +

A3C H66R E68Q C98S C101S

Vif.SIV

virions cells

CC-SS

A3C Tubulin A3C

A3C active site mutants are efficiently incorporated, but lose their activity

p27 mock vector

• nly

A3C H66R E68Q C98S C101S CC-SS

1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 luciferase activity [cps]

+ Vif Δ Vif

SIVagm ∆vif luc

Zn2+ co-ordinating residues in blue; non conserved residue which may need to be repaired are in red

His- 66 Cys-100 Cys- 97 Glu-68 A3C-Z2 A3F Ser Asp Glu Pro

Different activities of A3F and A3C against HIV-1: Key may be Glutamic acid in the helix and/or Proline in the turn

A3C A3F

HIV-1 (3-Plasmid Vector) HIV-1 (3-Plasmid Vector) SIVagm Luc Δ vif SIVagm Luc Δ vif

S61P mutation in A3C enhances the antiviral activity

CCCA and CCCG are specific for A3G, A3C may require others

A3C S61P shows slightly more in vitro cytidine deamination

CCUA ctrl GST A3G WT D99E S61P EP ctrl GST A3G WT D99E S61P EP

CCCA CCCG

A3C A3C ss DNA ds DNA digestion

qPCR shows a delay in the appearance of late RT-products

150 300 450 600 750 2 4 9 24 48 96

time post infection [hrs]

mock SIVΔvif only SIVΔvif + A3C

fg in 100ng gen. DNA R U5 U3

RNA Reverse Transcription

R R U5 U3

gag

late RT-products R R R R U5 U3

Die Integrase von HIV-1 als weiterer Antagonist von A3C?

mock SIV-IN HIV-IN

Lysat α-His

mock SIV-IN HIV-IN

Ni-NTA α-HA

mock SIV-IN HIV-IN Lysat

bound to

Lysat Lysat bound bound

A3C Vpr.A3C α-HA α-His HIV-IN

20 40 60 80 100 A3C Vpr.A3C

• rel. Binding

to HIV-IN [%]

Integrase of HIV-1 binds strongly to A3C

Protein-protein-interaction of A3C with integrases from SIV and HIV

DNA editing RNA editing

modified from Stevenson, Nature Medicine 2003

Important open questions:

• Why is human A3C not antiviral against HIV-1 (at least lab strains)?
• Why is sooty mangabey A3C antiviral against HIV-1?
• What is the antiviral activity of human A3C against SIV?
• Does Vif /A3 binding cause structural changes in either protein (Vif/A3 interface)?

Impacts oligomerization?

Klinik für Gastroenterologie, Hepatologie und Infektiologie Leber- und Infektionszentrum

• W. Hirsch-Institut für Tropenmedizin, ET

Direktor der Klinik Univ.-Prof. Dr. D. Häussinger

Virus Intrinsic Immune System Cancer Host-Virus Arms Races

Experimental results shown from:

Qinyong Gu Henning Hofmann Ananda Ayyappan Jaguva Vasudevan Zeli Zhang Jörg Zielonka

HIV only mock

A3C Vpr14-88-A3C + vif Δvif

• rel. luciferase activity [cps]

Fusion of a truncated VPR to A3C

100 101 102 103 104 105 106 α-HA α-Tubulin

HIV-1