The impact of APOBEC on the tropism of HIV Eva Heger AREVIR - PowerPoint PPT Presentation

The impact of APOBEC on the tropism of HIV Eva Heger AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Tropism – Coreceptor usage of HIV attachment to CD4 Coreceptor binding fusion gp41 gp120 V3 loop CD4 Coreceptor Chemokine receptor CCR5 or CXCR4 R5 X4 HIV AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

HIV infection Viruses with different tropism: • in early and chronical phase:  CCR5-tropic viruses (R5 virus) • 30-50% of the patients show coreceptor switch:  CXCR4-tropic viruses (X4 virus) CCR CXCR4 5 AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Tropism most important region gp41 CD4 gp120 V3 loop Coreceptor • third variable Region (V3) of gp120 (Choe et al., 1996) • aa 11/24/25 tropism relevant positions (Cardozo et al., 2007) amino acid sequence of V3 region (HXB2) CTRPNNNTRK S IHIGPGRAFYAT GE IIGDIRQAHC R5 virus D(Asp)/ E(Glu)/ G(Gly) HIV-Env-V3 GAN or GGN X4 virus R(Arg)/ K(Lys) HIV-Env-V3 AAR or AGR Deamination HIV (-)cDNA APOBEC Uracil Cytosin III II G A AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC proteins cellular antiviral proteins • C to U deamination in single ssDNA • G to A mutation in proviral DNA • APOBEC3G and APOBEC3F HIV protein - Vif viral counteracter  mediates the proteasomal degradation of APOBEC • AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC - Vif APOBEC3G and APOBEC3F no G-to-A mutation in proviral DNA • Vif A3G/F no Vif • no Degradation of A3G/A3F Replication G-to-A Hypermutation AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC - Vif APOBEC3G and APOBEC3F no G-to-A mutation in proviral DNA • Vif A3G/F no Vif • no Degradation of A3G/A3F Replication G-to-A Hypermutation AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC - Vif APOBEC3G und APOBEC3F G-to-A mutation in proviral DNA • A3G/F Vif Vif  viral infectivity factor • Degradation of A3G/A3F Replication AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC - Vif APOBEC3G und APOBEC3F G-to-A mutation in proviral DNA • A3G/F Vif Vif deficient variants • partial Degradation of A3G/A3F AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC - Vif APOBEC3G und APOBEC3F G-zu-A mutation in proviral DNA • Vif A3G/F Vif deficient variants • partial Degradation of A3G/A3F Replikation G-to-A Mutation AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Hypothesis Vif G-to-A mutations R5 Virus X4 Virus A3G/ A3F Aim ... mutagenic impact of APOBEC and the modulating effect of Vif upon the tropism of HIV AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC – Vif cell culture systems HI V APOBEC-expressing cells CXCR4/ Replication Infection CCR5 APOBEC NGS* analysis of newly produced virus population * Next generation sequencing AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC-cell culture systems Endogenous A3F and A3G Tet inducible cell lines A3G A3F Cells: PBMCs APOBEC3G APOBEC3F APOBEC3G APOBEC3F Tet/Dox Tet/Dox AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC inducible TetON system Tet inducible cell lines A3G A3F APOBEC3G APOBEC3F Tet/Dox Tet/Dox DG9 DF5  controlled expression of A3G and A3F  separate analysis of A3G and A3F activities AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC cell culture systems Endogenous A3F and A3G Cells: PBMCs APOBEC3G APOBEC3F AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Endogenous APOBEC system Endogenous A3F and A3G Cells: PBMCs APOBEC mRNA Level APOBEC3G APOBEC3F PBMCs 1 PBMCs 2 PBMCs 3 • Isolation of primary PBMCs from 3 HIV negative individuals • Determination of APOBEC mRNA levels: • unstimulated, 3 days after PHA stimulation and 5 days after HIV infection  increasing A3G/A3F mRNA levels after PHA stimulation  reduced A3G/A3F mRNA levels after HIV infection  PBMC cultures: individual different A3G and A3F levels AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Vif - APOBEC cell culture systems HI V APOBEC-expressing cells CXCR4/ Replication Infection CCR5 APOBEC NGS* analysis of newly produced virus population * Next generation sequencing AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Vif variants and APOBEC Activity against APOBEC APOBEC activity in the cell HI Vvif Mut R5 •WT - + •low - + •high •Del-Vif •intermediate - + •K22E •intermediate •Y40H - + (Simon V. et al., 2005) AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Workflow: 2. Infection wash Supernatant 2 days 1 2 3 4 5 6 7 1. Infection wash Supernatant 1 • • Tet inducible Tet inducible A3G and A3F A3G and A3F • PBMC • PBMC Viral load (p24 Ag) Infection + Next generation sequencing APOBEC inducible system analysis Cells: Tet inducible A3F/A3G cells HI Vvif Mut R5 •WT •Del-Vif •K22E, Y40H AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Impact of A3G and A3F on the replication of the Vif variants: APOBEC inducible system WT p24 Ag 1. Infection p24 Ag 2. Infection + ++ + ++ neg AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Impact of A3G and A3F on the replication of the Vif variants: APOBEC inducible system Del-Vif WT K22E + ++ + ++ + ++ + ++ + ++ + ++ neg neg neg p24 Ag 1. Infektion p24 Ag 2. Infektion  APOBEC mediated inhibition of HI V replication depends on Vif activity AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Impact of A3G and A3F on the replication of the Vif variants: APOBEC inducible system Del-Vif WT K22E + ++ + ++ + ++ + ++ + ++ + ++ neg neg neg NGS analysis amplification of ENV and NEF region env nef 900 bp 1.100 bp AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

NGS analysis: APOBEC inducible system env nef ENV amplicon 900 bp 20.000 Reads V3 region Geno2pheno [coreceptor] Classification of the V3 variants with FPR-Cutoff 10% R5 X4 / AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Proprotion of X4 variants: APOBEC inducible system + ++ + ++ neg AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Proprotion of X4 variants: APOBEC inducible system  X4-increment under APOBEC expression in the Vif- + ++ + ++ neg deficient variants AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Endogenous APOBEC cell culture system Endogenous A3F and A3G Cells: PBMCs APOBEC3G APOBEC3F AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Impact of A3G and A3F on the replication of the Vif variants: endogenous APOBEC system Endogenous A3F and A3G Cells: PBMCs APOBEC level APOBEC3G APOBEC3F PBMCs 1 PBMCs 2 PBMCs 3 Viral Load  APOBEC mediated inhibition of HIV replication depends on Vif activity  the lower the APOBEC level, the higher Viral Load AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Proprotion of X4 variants: APOBEC endogenous system  X4-increment depending on the Vif-deficiency AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

Calculation of mutation rates using NGS analysis: APOBEC endogenous system Amplicon of ENV and NEF-Region env nef 900 bp 1.100 bp about 20.000 reads ENV NEF G/A-Nucleotid frequencies at APOBEC motifs GG and GA  G-to-A mutation rates (% G-to-A mutation/position) AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC induced G-to-A mutation rates: endogenous system • Comparison of G-to-A mutation rates per position (% G-to-A mutation/position) • APOBEC motif (A3G/A3F) • region (ENV/NEF) • Vif variants low high AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC induced G-to-A mutation rates: endogenous system • Vif variants Increase with Vif deficiency AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC induced G-to-A mutation rates: endogenous system • Motifs Increase with Vif deficiency A3G: higher mutagenic activity AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -

APOBEC induced G-to-A mutation rates: endogenous system • Regions Increase with Vif deficiency A3G: higher mutagenic activity Nef: longer APOBEC-exposure AREVIR ∣ 09.05.2015 ∣ Eva Heger - The impact of APOBEC on the tropism of HIV -