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

▶
the impact of apobec on the tropism of hiv
SMART_READER_LITE
LIVE PREVIEW

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

Mar 22, 2024 416 likes •786 views

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

slide-1
SLIDE 1

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

The impact of APOBEC on the tropism of HIV

Eva Heger

slide-2
SLIDE 2

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Tropism – Coreceptor usage of HIV

gp41 gp120 V3 loop CD4 Coreceptor

Chemokine receptor CCR5 or CXCR4

R5 X4 HIV

attachment to CD4 Coreceptor binding fusion

slide-3
SLIDE 3

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 5 CXCR4

slide-4
SLIDE 4

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

amino acid sequence of V3 region (HXB2)

CTRPNNNTRKSIHIGPGRAFYATGEIIGDIRQAHC

R5 virus D(Asp)/ E(Glu)/ G(Gly)

HIV-Env-V3 HIV-Env-V3

R(Arg)/ K(Lys) X4 virus GAN or GGN AAR or AGR

HIV (-)cDNA Cytosin Uracil

Deamination

G A APOBEC III II

Tropism most important region

gp41 gp120 V3 loop CD4 Coreceptor

  • third variable Region (V3) of gp120 (Choe et al., 1996)
  • aa 11/24/25 tropism relevant positions (Cardozo et al., 2007)
slide-5
SLIDE 5

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
slide-6
SLIDE 6

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

APOBEC - Vif

APOBEC3G and APOBEC3F

  • G-to-A mutation in proviral DNA

A3G/F

Replication

no Vif

  • no Degradation of A3G/A3F

G-to-A Hypermutation

no Vif

slide-7
SLIDE 7

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

APOBEC - Vif

APOBEC3G and APOBEC3F

  • G-to-A mutation in proviral DNA

A3G/F

Replication

no Vif

  • no Degradation of A3G/A3F

G-to-A Hypermutation

no Vif

slide-8
SLIDE 8

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

Replication

Vif  viral infectivity factor

  • Degradation of A3G/A3F

Vif

A3G/F

slide-9
SLIDE 9

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

Vif deficient variants

  • partial Degradation of A3G/A3F

Vif

A3G/F

slide-10
SLIDE 10

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

Replikation

Vif deficient variants

  • partial Degradation of A3G/A3F

Vif

A3G/F

G-to-A Mutation

slide-11
SLIDE 11

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Aim

... mutagenic impact of APOBEC and the modulating effect of Vif upon the tropism of HIV

Hypothesis

Vif A3G/ A3F

R5 Virus X4 Virus

G-to-A mutations

slide-12
SLIDE 12

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

APOBEC – Vif cell culture systems

Infection

CXCR4/ CCR5

HI V NGS* analysis of newly produced virus population

APOBEC

* Next generation sequencing

Replication

APOBEC-expressing cells

slide-13
SLIDE 13

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

APOBEC-cell culture systems

Tet inducible cell lines A3G A3F Endogenous A3F and A3G

Cells: PBMCs

Tet/Dox

APOBEC3F

Tet/Dox

APOBEC3G APOBEC3F APOBEC3G

slide-14
SLIDE 14

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

Tet/Dox

APOBEC3F

Tet/Dox

APOBEC3G

DG9 DF5

 controlled expression of A3G and A3F  separate analysis of A3G and A3F activities

slide-15
SLIDE 15

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

APOBEC3F APOBEC3G

slide-16
SLIDE 16

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Endogenous APOBEC system

PBMCs 1 PBMCs 2 PBMCs 3 APOBEC mRNA Level

  • 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

 PBMC cultures: individual different A3G and A3F levels

Endogenous A3F and A3G

Cells: PBMCs APOBEC3F APOBEC3G

 increasing A3G/A3F mRNA levels after PHA stimulation  reduced A3G/A3F mRNA levels after HIV infection

slide-17
SLIDE 17

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Vif - APOBEC cell culture systems

Infection

CXCR4/ CCR5

HI V NGS* analysis of newly produced virus population

APOBEC

* Next generation sequencing

Replication

APOBEC-expressing cells

slide-18
SLIDE 18

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Vif variants and APOBEC

HI VvifMutR5 •WT

  • Del-Vif
  • K22E
  • Y40H

Activity against APOBEC

  • +
  • +
  • +
  • +

APOBEC activity in the cell

  • low
  • high
  • intermediate
  • intermediate

(Simon V. et al., 2005)

slide-19
SLIDE 19

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Workflow:

  • 1. Infection
  • 2. Infection

wash wash Supernatant 1 Supernatant 2

days 1 2 3 4 5 6 7 Infection

  • Tet inducible

A3G and A3F

  • PBMC

HI VvifMutR5 •WT

  • Del-Vif
  • K22E, Y40H

Viral load (p24 Ag) + Next generation sequencing analysis

  • Tet inducible

A3G and A3F

  • PBMC

APOBEC inducible system

Cells: Tet inducible A3F/A3G cells

slide-20
SLIDE 20

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

p24 Ag 1. Infection

p24 Ag 2. Infection

WT

++ + ++ +

neg

slide-21
SLIDE 21

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

p24 Ag 1. Infektion

p24 Ag 2. Infektion

WT Del-Vif K22E

 APOBEC mediated inhibition of HI V replication depends on Vif activity

Impact of A3G and A3F on the replication of the Vif variants: APOBEC inducible system

++ + ++ + ++ + ++ +

neg neg

++ + ++ +

neg

slide-22
SLIDE 22

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

WT Del-Vif K22E

Impact of A3G and A3F on the replication of the Vif variants: APOBEC inducible system

++ + ++ + ++ + ++ +

neg neg

++ + ++ +

neg

NGS analysis

1.100 bp 900 bp

env nef

amplification of ENV and NEF region

slide-23
SLIDE 23

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

NGS analysis: APOBEC inducible system

900 bp

env nef

ENV amplicon

V3 region Geno2pheno[coreceptor]

Classification of the V3 variants with FPR-Cutoff 10%

R5 X4

/

20.000 Reads

slide-24
SLIDE 24

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Proprotion of X4 variants: APOBEC inducible system

++ + ++ +

neg

slide-25
SLIDE 25

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Proprotion of X4 variants: APOBEC inducible system

++ + ++ +

neg

 X4-increment under APOBEC expression in the Vif- deficient variants

slide-26
SLIDE 26

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

APOBEC3F APOBEC3G

slide-27
SLIDE 27

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

APOBEC level

Impact of A3G and A3F on the replication of the Vif variants: endogenous APOBEC system

PBMCs 1 PBMCs 2 PBMCs 3

 APOBEC mediated inhibition of HIV replication depends on Vif activity  the lower the APOBEC level, the higher Viral Load

Endogenous A3F and A3G

Cells: PBMCs APOBEC3F APOBEC3G

Viral Load

slide-28
SLIDE 28

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

slide-29
SLIDE 29

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

1.100 bp 900 bp

env nef

Amplicon of ENV and NEF-Region ENV NEF G/A-Nucleotid frequencies at APOBEC motifs GG and GA  G-to-A mutation rates (% G-to-A mutation/position)

about 20.000 reads

slide-30
SLIDE 30

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

APOBEC induced G-to-A mutation rates: endogenous system

low high

  • Comparison of G-to-A mutation rates per position (% G-to-A mutation/position)
  • APOBEC motif (A3G/A3F)
  • region (ENV/NEF)
  • Vif variants
slide-31
SLIDE 31

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

slide-32
SLIDE 32

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -
  • Motifs

A3G: higher mutagenic activity Increase with Vif deficiency

APOBEC induced G-to-A mutation rates: endogenous system

slide-33
SLIDE 33

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -
  • Regions

A3G: higher mutagenic activity Nef: longer APOBEC-exposure Increase with Vif deficiency

APOBEC induced G-to-A mutation rates: endogenous system

slide-34
SLIDE 34

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Results - Summary

... APOBEC-Vif-Interaction was confirmed in the inducible and endogenous cell culture system ...A3G and A3F as mutagenic factors for the emergence of X4 variants identified ...Vif modulates APOBEC activity increasing mutation rates

Hypothesis

Vif A3G/ A3F

R5 virus X4 virus

G-to-A mutations

slide-35
SLIDE 35

AREVIR ∣09.05.2015 ∣ Eva Heger

  • The impact of APOBEC on the tropism of HIV -

Thank you for your attention!

and

Rolf Kaiser

Alex Thielen, Martin Däumer

Institute of Immunology and Genetics, Labor Dr. Thiele, Kaiserslautern