HIV’s interaction with APOBEC3C (a work in progress) Carsten Münk Clinic for Gastroenterology, Hepatology and Infectiology Heinrich-Heine-University Düsseldorf
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 ssDNA zinc atom hydrophobic five –stranded β sheets ( β 1- β 5) surrounded by six α helices ( α 1- α 6) Jaguva Vasudevan et. al 2013
Vif binding to APOBEC3s (A3) required for A3 proteasomal degradation A3 Vif A3 degradation Aydin et al. 2014
Encapsidated APOBEC3G deaminates deoxy-cytidines in single stranded viral DNA Cytidin-Deamination of ssDNA Mutations ! Münk et al. 2012
A3G binds the HIV RNA genome, enzymatic activity on ssDNA Chaurasiya et al. 2013
Encapsidated APOBEC3G deaminates deoxy-cytidines in single stranded viral DNA PCR, 10 h post infection HIV-1 wt HIV-1 Δ vif each line represents an individual PCR on viral DNA
Nucleotide preferences of human A3s in virus infection Trinucleotide preferences CCC TCC ACC TTC ATC TTC ATC DÖRRSCHUCK ET AL. 2011
A3G binds ssDNA mainly as a dimer Jaguva Vasudevan et. al 2013 RNA interaction High Molecular Mass complexes of A3G
A3G monomers/dimers convert to oligomers that slowly dissociate Chaurasiya et al. 2013
APOBEC3G: 3 ′→5′ ‘slide and jump’ mechanism, no random ‘on and off’ mechanism Chelico et al. 2006
APOBEC3s strongly restrict cross-species lentiviral transmissions between distantly related mammals FIV (or Bovine immunodeficiency virus BIV in cattle, Equine infectious anemia virus EIAV in horses/donkeys, Visna in sheep) HIV
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 + E3 Ligase A3 degradation
APOBEC3C is Z2 protein active domain
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.
Species-specific Vif A3C/A3Z2 interactions rhe A3C FeA3Z2b AGM A3C SmmA3C Vif-V5 CPZ A3C α Tubulin hu A3C α Tubulin HIV-1 Vif C1 C4 C5 C30 Vif-V5 α Tubulin
Chimeric A3C/Z2 (feline/human): 46% ident. AA huA3C feA3Z2b huA3C feA3Z2b huA3C Vif binding pocket Kitamura et al. 2012 feA3Z2b HIV-1 Vif HIV-1 Vif induced degradation: + - + - - -
human : mangabey A3C: 76% ident. AA Human A3C Mangab. A3C Human A3C Mangab. A3C Vif binding pocket Kitamura et al. 2012 Human A3C Mangab. A3C HIV-1 Vif induced degradation: + HuA3C - SmmA3C C1 C2 C3 - + - + - + - + HIV1 VIF C4 C5 normal exp. 5sec + Anti-HA C6 C7 over exp. 30sec - C8 Anti-v5 C9 tubulin - C10
Transfection human A3C , rhesus A3C and their chimers with HIV1 VIF HuA3C 85% ident. AA RhA3C RhehuA3C9 RhehuA3C11 RhehuA3C13 RhehuA3C15 RhehuA3C17 RhehuA3C31 HIV1 VIF - + - + - + - + - + - + - + - + Anti-HA Anti-v5 tubulin 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.
1 Human A3C Rhesus A3C 3 4 5 6 Human A3C Rhesus A3C Vif binding pocket Kitamura et al. 2012 Human A3C Rhesus A3C 85% ident. AA HIV-1 Vif induced 6 degradation: 1 3 4 5 HuA3C + RhA3C - RhehuA3C9 + RhehuA3C11 + RhehuA3C13 - RhehuA3C15 - RhehuA3C17 - RhehuA3C31 -
HIV-1 Vif binding region in A3C: extended view Looks like residues both are forming a cavity where vif binds! Vif binding surface (Kitamara et al.) New extended Vif binding surface 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
HIV-1 Vif binding domains 1 94 VIF B-NL4-3 W DRMR W SLVK F H YRHHY V IPL L Y L TGER W LG G I W A3F A3G A3G A3H A3F/A3G A3F/A3G A3F A3F/A3G WX2DRMR WXSLVK YRHHY FX8H VXIPLX4L LGXGX2IXW YXXL TGERXW APOBEC3 N-Term binding 95 193 EDRW S LQYLA PPLP L TQ AD I H C C H A3F/A3G A3F BC BOX/CuL5-E3 Cul5-E3 Cul5-E3(HCCH) TQX5ADX2I (EDRW) SLQYLA (PPLPX4L) C-Term E3 ligase binding
HIV-1 Vif binding domains A3C ? R R 1 94 VIF W DRMR W SLVK F H YRHHY B-NL4-3 V IPL L Y L TGER W LG G I W A3F A3G A3G A3H A3F/A3G A3F/A3G A3F A3F/A3G WX2DRMR WXSLVK YRHHY FX8H VXIPLX4L LGXGX2IXW YXXL TGERXW N-Term APOBEC3 binding A3C ? E D 95 193 EDRW S LQYLA PPLP L TQ AD I H C C H A3F/A3G A3F BC BOX/CuL5-E3 Cul5-E3 Cul5-E3(HCCH) TQX5ADX2I (EDRW) SLQYLA (PPLPX4L) C-Term E3 ligase binding
Determinants in HIV-1 and HIV-2 Vif important for A3C degradation HIV-1 Vif structure A3C Vif-V5 α Tubulin red color: RR and ED residues in HIV-1 Vif
Vif HIV-1 Plasmids in the lab A-R37 A-008 B-NL4-3 B-LAI subtype B-026 B-R01 B B-102# B-JRCSF C-564 Group C-748 M 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)
A3C degradation: some Vifs are different A3C A3G B B D E F F B B D E F F - - - Vif: NL4-3 R01 158 402 029 019 NL4-3 R01 158 402 029 019 A3G A3C Vif GAPDH
A3C degradation: B-NL4-3: + D-158: ++ F-029: -- A3C R R 1 B-NL4-3 D-158 F-029 W DRMR W SLVK F H YRHHY V IPL L Y L TGER W LG G I W A3F A3G A3G A3H A3F/A3G A3F/A3G A3F A3F/A3G WX2DRMR WXSLVK YRHHY FX8H VXIPLX4L LGXGX2IXW YXXL TGERXW A3C E D 193 B-NL4-3 D-158 F-029 EDRW S LQYLA PPLP L TQ AD I H C C H A3F/A3G A3F BC BOX/CuL5-E3 Cul5-E3 Cul5-E3(HCCH) TQX5ADX2I (EDRW) SLQYLA (PPLPX4L) more complex than predicted
A3C is not antiviral against HIV-1 NL4-3, but strongly inhibits SIVagm Δ Vif HIV-1 ∆vif luc SIVagm ∆vif luc 1 0 5 + Vif ∆ Vif 1 0 4 rel luciferase activity [cps] 1 0 3 1 0 2 1 0 1 1 0 0 HIV only A3C A3G SIV only A3C A3G A3C A3G A3C A3G + - + - + - + - Vif APOBEC3 cells α -Tub APOBEC3 virions α -CA HIV-1 ∆vif luc SIVagm ∆vif luc
Why SIV, not HIV-1? Sub-viral localisation of A3C? SIV HIV-1 A3C A3G
Will core localizing HIV-1 Vpr domain increase Hypothese I the anti HIV-activity of A3C? Zn 2+ NH 3 A3C COOH Zn 2+ NH 3 Vpr.A3C COOH Gly 4 -Ser SIV HIV1 Vpr.A3C A3C
Vpr-A3C: more potent and Vif-resistant Vpr.3C inhibiert HIV Vif-unabhängig rel. luciferase activity (cps) Vpr-A3C Vpr-A3C + Vif µg plasmd DNA Vpr-A3C + Vif Vpr-A3C
Purification of HIV-1 Cores viral glycoprotein (VSV-G) optiprep viral core fraction: IN, RT, capsid (p24)
Both A3C and Vpr.A3C localize to cores HIV ∆vif + Vpr.3C HIV ∆vif + A3C Density Density RT Aktivität Dichte RT activity (pg/ml) RT Aktivität Dichte Density Density RT Aktivität [pg/mL] RT Aktivität [pg/mL] 8000 1,4 10000 1,4 RT activity (pg/ml) Dichte ρ [g/mL] Dichte ρ [g/mL] 8000 1,3 6000 1,3 6000 4000 1,2 1,2 4000 1,1 2000 1,1 2000 0 1 0 1 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 Fraktion # Fraktion # Fraction # Fraction # HIV ∆vif + Vpr.3C HIV ∆vif + A3C Fraction Fraction 1 2 3 4 5 6 7 8 9 10 11 Fraktion # Fraktion # 1 2 3 4 5 6 7 8 9 10 11 α -HA α -HA α -VSV-G α -VSV-G % OptiPrep 60 % OptiPrep 20 60 20
A3C determinants required for anti-HIV-1 activity huA3G huA3C CPZA3C rhA3C AGM A3C SmmA3C FcaA3Z2b pcDNA3.1 pcDNA3.1 pcDNA3.1 pcDNA3.1 pcDNA3.1 pcDNA3.1 pcDNA3.1 10ng 150ng 10ng 150ng 10ng 150ng 10ng 150ng 10ng 150ng 10ng 150ng 10ng 150ng α -HA α -Tubulin HIV-1 Luc (3 plasmid system)
human : mangabey A3C: 76% ident. AA Human A3C Mangab. A3C Human A3C Mangab. A3C Human A3C Mangab. A3C HuA3C SmmA3C C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
A3C and SIVagm: no induction of G-to-A mutations SIVagm Δ vif +/- APOBEC3 (DNaseI treated) HOS Isolation of total DNA PCR (Taq Pol) Sequencing G A mutations?
Is the Zn 2+ -finger the active domain? A3G-C term (Z1) A3C (Z2) ? His-66 His-257 Cys-97 Zn 2+ Cys-288 Zn 2+ Glu-68 Cys-100 Cys-291 Glu-256 A3C A3G active site 2 active site active site 1 NH 3 NH 3 COOH COOH Zn 2+ HxE------PCxxC HxE------PCxxC A3Cwt: H-X-E-X 27 -P-C-X 2 -C incorporation in H66R: R-X-E-X 27 -P-C-X 2 -C catalytic the virion active site E68Q: H-X-Q-X 27 -P-C-X 2 -C C98S: H-X-E-X 27 -P-S-X 2 -C C101S: H-X-E-X 27 -P-C-X 2 -S CC-SS: H-X-E-X 27 -P-S-X 2 -S
Recommend
More recommend
