HIV-1's different ways to survive against hostile APOBEC3 proteins - - PowerPoint PPT Presentation
HIV-1's different ways to survive against hostile APOBEC3 proteins Carsten Mnk Klinik fr Gastroenterologie, Hepatologie und Infektiologie Universittsklinikum Dsseldorf HIV Replication: HIV Replication: some cellular proteins are
Carsten Münk Klinik für Gastroenterologie, Hepatologie und Infektiologie Universitätsklinikum Düsseldorf
CD4 CD4 CCR5 CCR5
TRIM5α TRIM5α Tetherin Tetherin APOBEC3 APOBEC3
Background on APOBEC3 genes and proteins Three ways
APOBEC3s Clinical relevance
Wissing et al. 2010, modified
Vif
Wissing et al. 2010, modified
His‐Xaa‐Glu‐Xaa23‐28 ‐Pro‐Cys‐Xaa2‐4 ‐Cys
100 AS APOBEC1 APOBEC2 APOBEC3A APOBEC3B APOBEC3C APOBEC3D APOBEC3F APOBEC3G APOBEC3H APOBEC4 AID
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
Z2 Z3 Z1
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
Thr Thr Tyr Tyr Leu Leu Leu Leu ACC TA ACC TAT T TTG CTG TTG CTG ACC TA ACC TAC C TTG CTG TTG CTG Thr Thr Tyr Tyr Leu Leu Leu Leu Thr Thr Tyr Tyr Leu Leu Leu Leu ACC T ACC TA AT TTG CTG T TTG CTG ACC T ACC TC CT TTG CTG T TTG CTG Thr Thr Ser Ser Leu Leu Leu Leu
all sequences
Münk and Bravo 2011, submitted
position protein sequence
primate sequences
position protein sequence
TTC ATC TTC ATC CCC TCC ACC no A3 huA3G huA3F huA3B
nucleotide position nucleotide position Dörrschuck….. Münk, Tönjes 2011
TET proteins: a group of Fe(II)/2-oxoglutarate-dependent dioxygenases SMUG1: single-strand-selective monofunctional uracil-DNA glycosylase 1 TDG: thymine-DNA glycosylase
base excision repair DNA methyltransferases Cytosine 5-Methylcytosine 5-Hydoxy- Methylcytosine 5-Hydoxy- Methyluracil
Guo et al. Cell 2011
Hepatitis B virus
Turelli et al. 2004; Suspene et al. 2005 Rösler et al. 2005; Bonvin et al. 2006 Baumert et al. 2007; Jost et al. 2007 Vartanian et al. 2010
Para- Retrovirus
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
Papillomavirus
Vartanian et al. 2008
DNA-Virus
Hepatis C virus
Peng et al. 2011
Paramyxoviruses (measles, mumps…)
Fehrholz et al. POSTER Freiburg 2011
RNA-Virus
WNV: West Nil virus VEEV: Venezuelan equine encephalitis virus
Schoggins et al. Nature 2011
Dot plots of large-scale ISG screens against six viruses.
20 40 60 80 100 120 A3A A3B A3C A3D A3F A3G A3H no A3 % Infectivity HIV-1 ΔVif HIV-1 wt
Infectivity
APOBEC3 expression in naıve and stimulated CD4+ lymphocytes
A3C high expressed in T cells
HIV-1 „escapes“ A3B by avoiding replication in B-cells!
Eric W. Refsland et al., Nucleic Acids Research, 2010
HIV-1- Luc
1 10 102 103 104 105 106
no virus vector
A3C Luciferase activity [cps]
wt Δ vif
SIVagm-Luc
1 10 102 103 104 105
no virus vector
A3C Luciferase activity [cps]
wt Δ vif
luciferase activity [cps]
A3 C A3 G + - + - HI V-1 A3 C A3 G virions cell lysates
A3 A3 p24/27 Tubulin
+ - + - SI V agm
1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 vector
A3C A3G A3C A3G
+ Vif Δ Vif Vif HI V-1 SI V agm
GST GST GST GST NC HIV1 NC SIV NC HIV1 NC SIV lysate A3C-IP GST GST NC HIV1 NC SIV A3C-IP αGST (Nucleocapsid) αHA (APOBEC3C)
Nucleocapsid
pET SIV HIV-1 HIV-2 lysate pET SIV HIV-1 HIV-2 IP anti-His (Integrase) pET SIV HIV-1 HIV-2 IP input αHA (APOBEC3C)
Integrase
HIV-1
VPR
20 40 60 80 100 120 140
0.04 0.268 0.8 1.2 µg Vector
% infectivity
VPR-A3C -vif VPR-A3C +vif A3C - vif A3C + vif
VPR
A3C VPR-A3C
transfected A3C-DNA
Hofmann, H and C. Münk, in preparation
Hofmann, H and C. Münk, in preparation
Next steps:
the A3C inhibiting mechanism
why Vpr-fusion blocks the Vif induced degradation
Vif APOBEC3G Inhibitor
Vif
VifHIV : no in vivo phosphorylation in 293T cells, no phosphorylation in vitro by ERK2. VifHIV : no in vivo phosphorylation in 293T cells, no phosphorylation in vitro by ERK2. In vivo 32P labeling In vitro 33P kinase assay
Kopietz, F. and C. Münk, in revision
native condition +5% coomasie SDS+heat
20mer Oligomeric forms (~6,10,14,20,30) of denaturated multimer Dimer& trimer
Multimer Dimer salts Peak 1(large) Peak 2 (small) Distorted Zn peak Peak 1(large) Peak 2 (small) Distorted Zn peak Peak 1(large) Peak 2 (small) Distorted Zn peak M 480 242 146 66 720 1048 1236 20 native condition
Size exclusion chromatography Vif: ~23 kDa, rVif-His ~ 24 kDa
kDa
Stauch B, […], Münk C, Schneider G., PNAS 2009
A3C dimer A3C monomer
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
for disease progression?
Study Selected relationships reported
Pace et al. 2006 Gandhi et al. 2008 Land et al. 2008 Vazquez-Perez et al. 2009 Ulenga et al. 2008 Piantadosi et al. 2009 Reduced viraemia attributable to hypermutation Hypermutation comparable groups, exception
elite suppressor who had significant elevated hypermutation levels. Increasd CD4+ cell counts in 17 patients with significant hypermutation. Increased hypermutation in patients with low viral load No correlation between hypermutation and viremia No correlation between hypermutation and either viral load in chronic infection
CD4+ cell counts
Correlation found
+
+
Study Selected relationships reported
Jin et al. 2005 Ulenga et al. 2008 Vazquez-Perez et al. 2009 Cho et al. 2006 Inverse correlation between A3G expression and viral
between A3G expression and CD4+ cell count among
A3G mRNA levels in LTNPs and uninfected individuals relative to progressors Inverse correlation between A3G or A3F mRNA levels after infection and viral set point. Positive correlation between A3G mRNA expression and CD4+ cell counts in infected
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
between A3F or A3G mRNA levels and viral load
CD4* cell counts
Inverse correlation to viral load found
+
+
Study Selected relationships reported
An et al. 2004 Valcke et al. 2008 Do et al. 2005 Pace et al. 2006 7 polymorphisms identified, H186R more common in African Americans and correlates with loss
and progression to AIDS 6 polymorphisms identified, C40693T intronic change associated with increased risk
infektion None
29 SNPs identified in A3G associated with rate of disease progression 22 polymorphism identified, no association found
+ +
found
An et al. 2004
Kim et al. JVi 2010 Mulder et al. PNAS 2008 Vif
I) Vif counteracts A3F, A3G II) Unknown resistance mechanism against A3C III) Tropism for T-cells/macrophages escape from A3B
I) Block binding Vif- A3F, A3G Enhance stability
Decrease stability
II) Block resistance to A3C III) Induce A3B in T-cells/macrophages
Klinik für Gastroenterologie, Hepatologie und Infektiologie
(Director: Univ.-Prof. Dr. D. Häussinger) Ananda Ayyappan J.V. Benjamin Gabriel Wioletta Hörschken Henning Hofmann Ferdinand Kopietz Aikatharina Krikoni Melanie Krämer Daniela Marino Mario Perković Jörg Zielonka
Collaborations:
Arndt Borckhardt, Heiner Schaal UKD, Düsseldorf Dieter WiIllbold, Lutz Schmitt HHU, Düsseldorf Klaus Cichutek, Egbert, Flory, Paul-Ehrlich-Institut Gerald Schumann, Ralf Tönjes, Paul-Ehrlich-Institut Martin Löchelt, DKFZ Ignacio G. Bravo, Barcelona Gisbert Schneider, ETH Zürich Stephen J. O‘Brien, NCI-Frederick Viviana Simon, Mt. Sinai Hospital, New York
Heinz-Ansmann Stiftung für AIDS Forschung
+ vif no vif
Visna BIV EIAV RELIK FIV pSIVgml SIV HI V
Lagomorph Group
endogenous viruses
exogenous viruses
Feline Group Equine Group Bovine Group Ovine- Caprine Group Primate Group