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 - + - + - + - +

over 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

100

101

102

103

104

105

A3C A3G A3C A3G SIV only HIV only

+ Vif ∆ Vif

HIV-1 ∆vif luc SIVagm ∆vif luc

rel l

ucife

rase

act

ivity

[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.

luci

fera

se a

ctiv

ity (c

ps)

viral glycoprotein (VSV-G)

viral core fraction: IN, RT, capsid (p24)

Purification of HIV-1 Cores

optiprep

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

0

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

Aktiv

ität [

pg/m

L] D

ichte 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

0 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

Aktiv

ität [

pg/m

L] D

ichte ρ [g/mL]

Fraktion #

Density

Density

Fraction Fraction

RT a

ctiv

ity (p

g/m

l)

RT a

ctiv

ity (p

g/m

l)

Density Density

Fraction # Fraction #

α-Tubulin

α-HA

huA3G huA3C CPZA3C 10ng 150ng

pcDN

A3.1

10ng 150ng

pcDN

A3.1

10ng 150ng

pcDN

A3.1

rhA3C AGM A3C 10ng 150ng

pcDN

A3.1

10ng 150ng

pcDN

A3.1

SmmA3C 10ng 150ng

pcDN

A3.1

FcaA3Z2b 10ng 150ng

pcDN

A3.1

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

GA 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 only

A3C H66R E68Q C98S C101S CC-SS 100

101

102

103

104

105

luci

fera

se a

ctiv

ity [c

ps]

+ 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

0

150

300

450

600

750

2 4 9 24 48 96

time post infection [hrs]

mock

SIVΔvif only

SIVΔvif + A3C

fg in

100

ng g

en. D

NA

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?

moc

k

SIV-

IN

HIV

-IN

Lysat

α-His

moc

k

SIV-

IN

HIV

-IN

Ni-NTA

α-HA

moc

k

SIV-

IN

HIV

-IN

Lysa

t

bound to

Lysa

t

Lysa

t

boun

d

boun

d

A3C Vpr.A3C

α-HA

α-His HIV-IN

0 20 40 60 80 100

A3C Vpr.A3C re

l. B

indi

ng

to H

IV-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

moc

k

A3C Vpr14-88-A3C

+ vif

Δvif

rel.

luci

fera

se a

ctiv

ity [c

ps]

Fusion of a truncated VPR to A3C

100

101

102

103

104

105

106

α-HA

α-Tubulin

HIV-1