HIV-1's different ways to survive against hostile APOBEC3 proteins Carsten Münk Klinik für Gastroenterologie, Hepatologie und Infektiologie Universitätsklinikum Düsseldorf
HIV Replication: HIV Replication: some cellular proteins are inhibitors some cellular proteins are inhibitors CD4 CD4 TRIM5 α TRIM5 α CCR5 CCR5 APOBEC3 APOBEC3 Tetherin Tetherin
Background on APOBEC3 genes and proteins Three ways of HIV ‐ 1 against APOBEC3s Clinical relevance
Model of HIV ‐ 1 ‐ APOBEC3 interaction Χ Wissing et al. 2010, modified
Model of HIV ‐ 1 ‐ APOBEC3 interaction Vif Wissing et al. 2010, modified
Human APOBEC3 Proteins 100 AS APOBEC1 AID APOBEC2 APOBEC3A APOBEC3B APOBEC3C APOBEC3D APOBEC3F APOBEC3G APOBEC3H APOBEC4 His ‐ Xaa ‐ Glu ‐ Xaa 23 ‐ 28 ‐ Pro ‐ Cys ‐ Xaa 2 ‐ 4 ‐ Cys
Something drove the A3 gene expansion during placental diversification Z2 Z3 Z1 Projection of the A3 phylogenetic relationships onto the human genomic locus. Labels indicate median age [95% Highest Posterior Density] of the corresponding nodes. Münk and Bravo 2011, submitted
A Ka/Ks ratio >1 indicates arms ‐ race Ka Ks Ka Ks Synonymous Non-synonymous substitution rate substitution rate Thr Tyr Leu Leu Leu Thr Tyr Leu Thr Tyr Leu Leu Leu Thr Tyr Leu ACC TA T ACC TA T TTG CTG TTG CTG ACC T A T TTG CTG ACC T A T TTG CTG ACC TA C C TTG CTG TTG CTG ACC TA ACC T C C T TTG CTG T TTG CTG ACC T Thr Tyr Leu Leu Leu Thr Tyr Leu Thr Ser Leu Leu Leu Thr Ser Leu Purifying selection: Ka/Ks < 1 e.g. histone genes Neutral selection: Ka/Ks = 1 pseudo-genes Positive selection: Ka/Ks > 1 host-pathogen arms-race
Slide ‐ window analysis of the ka/ks values of APOBEC3 genes primate sequences all sequences position protein sequence position protein sequence Münk and Bravo 2011, submitted
Nucleotide preferences of human A3 proteins huA3G CC C TC C AC C huA3F TT C AT C no A3 huA3B TT C nucleotide position AT C nucleotide position Dörrschuck….. Münk, Tönjes 2011
APOBEC3 proteins can deaminate 5hmC DNA 5-Methylcytosine Cytosine methyltransferases base excision repair 5-Hydoxy- 5-Hydoxy- Methylcytosine Methyluracil Guo et al. Cell 2011 TET proteins: a group of Fe(II)/2-oxoglutarate-dependent dioxygenases TDG: thymine-DNA glycosylase SMUG1: single-strand-selective monofunctional uracil-DNA glycosylase 1
Non ‐ retroviruses sensitive to APOBEC3s Hepatitis B virus Turelli et al. 2004; Suspene et al. 2005 Para- Rösler et al. 2005; Bonvin et al. 2006 Retrovirus Baumert et al. 2007; Jost et al. 2007 Vartanian et al. 2010 Parvoviruses (AAV-2) Chen et al. 2006; Zielonka et al. 2009 Narvaiza et al. 2009; Bulliard et al. 2011 TT-virus Tsuge et al. 2008 DNA-Virus Papillomavirus Vartanian et al. 2008 Hepatis C virus Peng et al. 2011 RNA-Virus Paramyxoviruses (measles, mumps…) Fehrholz et al. POSTER Freiburg 2011
APOBEC3A can enhance replication of WNV and VEEV Dot plots of large-scale ISG screens against six viruses. WNV : West Nil virus Schoggins et al. Nature 2011 VEEV : Venezuelan equine encephalitis virus
HIV ‐ 1: three types of A3 interaction Infectivity of HIV-1 120 HIV-1 wt HIV-1 Δ Vif 100 % Infectivity 80 60 40 20 0 A3G no A3 A3A A3B A3C A3D A3F A3H
APOBEC3B not expressed in HIV ‐ 1 target cells APOBEC3 expression in naıve and stimulated CD4 + lymphocytes Eric W. Refsland et al., Nucleic Acids Research, 2010 HIV-1 „escapes“ A3B by avoiding replication in B-cells! A3C high expressed in T cells
APOBEC3C inhibits Δ vif SIV HIV-1- Luc SIVagm-Luc 10 6 10 5 wt wt Δ Δ vif vif 10 5 10 4 Luciferase activity [cps] Luciferase activity [cps] 10 4 10 3 10 3 10 2 10 2 10 10 1 1 no vector A3C no vector A3C virus only virus only
APOBEC3C is packaged into HIV ‐ 1 particles SI V agm HI V-1 A3 G A3 C A3 C A3 G Vif + - + - + - + - A3 cell lysates Tubulin A3 virions p24/27 luciferase activity [cps] 1 0 5 + Vif 1 0 4 Δ Vif 1 0 3 1 0 2 1 0 1 1 0 0 vector A3C A3G A3C A3G only SI V agm HI V-1
Does A3C interacts differentially with HIV ‐ 1? Nucleocapsid Integrase lysate A3C-IP IP lysate NC HIV1 NC HIV1 NC SIV NC SIV HIV-1 HIV-2 HIV-1 HIV-2 pET pET SIV GST GST GST GST SIV anti-His α GST (Integrase) (Nucleocapsid) IP A3C-IP HIV-1 HIV-2 NC HIV1 input NC SIV pET SIV GST GST α HA (APOBEC3C) α HA (APOBEC3C)
Sub ‐ viral localisation of A3C? HIV-1 A3C VPR- VPR A3C
VPR fusion to A3C cracks resistance of HIV ‐ 1 A3C VPR-A3C A3C - vif VPR A3C + vif VPR-A3C -vif 140 120 VPR-A3C +vif 100 % infectivity 80 60 40 20 0 Vector 0.04 0.268 0.8 1.2 µg only transfected A3C-DNA Hofmann, H and C. Münk, in preparation
A3C and VPR.A3C both localize to the core of HIV Hofmann, H and C. Münk, in preparation
Next steps: -identify the A3C inhibiting mechanism -understand why Vpr-fusion blocks the Vif induced degradation
Vif bindung to APOBEC3G: a target for new antiviral drugs? Inhibitor APOBEC3G Vif Structures of Vif and APOBEC3G and their interaction not well known !
Is Vif activity regulated by phosphorylation? Vif
Search for Vif phosphorylation In vivo 32 P labeling In vitro 33 P kinase assay Vif HIV : no in vivo phosphorylation in 293T cells, no phosphorylation Vif HIV : no in vivo phosphorylation in 293T cells, no phosphorylation in vitro by ERK2. in vitro by ERK2. Kopietz, F. and C. Münk, in revision
Vif forms dimers and 20mers native condition native condition +5% coomasie SDS+heat Distorted Zn peak Distorted Zn peak Distorted Zn peak Peak 2 (small) Peak 2 (small) Peak 2 (small) Peak 1(large) Peak 1(large) Peak 1(large) kDa M Multimer 1236 1048 720 480 20mer 242 Dimer Oligomeric forms 146 salts (~6,10,14,20,30) of 66 denaturated multimer Dimer& trimer 20 Vif: ~23 kDa, rVif-His ~ 24 kDa Size exclusion chromatography
Some structural information available for A3s A3C dimer A3C monomer Stauch B, […], Münk C , Schneider G., PNAS 2009
Clinical relevance ? Vif does not completely prevent the A3 induced editing in vivo . Part of the viral variabilty is caused by A3s: Drug-resistence Immune-escape Rezeptor switch Variability of A3s: a factor for disease progression?
Linkage studies between clinical indicators and hypermutation Correlation found Study Selected relationships reported + Pace et al . Reduced viraemia attributable to hypermutation 2006 -/+ Gandhi et al. Hypermutation comparable groups, exception one elite suppressor who had 2008 significant elevated hypermutation levels. + Land et al. Increasd CD4+ cell counts in 17 patients with significant hypermutation. 2008 + Increased hypermutation in patients with Vazquez-Perez low viral load et al. 2009 No correlation between hypermutation and Ulenga et al. - viremia 2008 - No correlation between hypermutation and Piantadosi either viral load in chronic infection or et al. 2009 CD4+ cell counts
Linkage studies between clinical indicators + APOBEC3 expression Inverse correlation to Study Selected relationships reported viral load found Inverse correlation between A3G expression Jin et al . and viral load. Positive correlation between 2005 A3G expression and CD4+ cell count among + infected. Higher A3G mRNA levels in LTNPs and uninfected individuals relative to progressors Inverse correlation between A3G or A3F + Ulenga et al. mRNA levels after infection and viral set point. 2008 Positive correlation between A3G mRNA Vazquez-Perez expression and CD4+ cell counts in infected et al. 2009 + patients. Negative correlation between A3G mRNA expression and viraemia. Higher A3G mRNA expression in exposed seronegative individuals compare with healthy controls. Lower A3Fand A3G mRNA levels in infected Cho et al. vs. uninfected individuals. No correlation 2006 - between A3F or A3G mRNA levels and viral load or CD4* cell counts
Linkage studies between clinical indicators + APOBEC3 polymorphism Correlation found Study Selected relationships reported + An et al . 7 polymorphisms identified, H186R more common in African Americans and correlates 2004 with loss of CD4+ cells and progression to AIDS Valcke 6 polymorphisms identified, C40693T intronic + et al. 2008 change associated with increased risk of infektion - Do et al. None of the 29 SNPs identified in A3G 2005 associated with rate of disease progression - Pace 22 polymorphism identified, no association et al. 2006 found
Polymorphic in African Americans (AA) (f = 37%) and rare in European Americans (f < 3%) An et al. 2004
A3G ‐ mediated editing can accelerate adaptation to selective pressure Vif Mulder et al. PNAS 2008 Kim et al. JVi 2010
HIV ‐ 1: three viral mechanisms against human APOBEC3 proteins I) Vif counteracts A3F, A3G II) Unknown resistance mechanism against A3C III) Tropism for T-cells/macrophages escape from A3B
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