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Warner C' Greene MD, PhD

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Drug Development: The Vif-APOBEC3G Axis. Each of us in this room ... HSA (Mo CD24) ... HSA (Mo CD24) LMM APOBEC3G Restricts HIV-1 After Entry. into Resting CD4 T Cells ... – PowerPoint PPT presentation

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Title: Warner C' Greene MD, PhD


1
Cell-Virus Interactions as Targets for Drug
Development The Vif-APOBEC3G Axis
Warner C. Greene MD, PhD Director and Senior
Investigator
2
Each of us in this roomproduces a potent innate
antiviral factorthat can potentially stop
HIVdead in its tracks
  • APOBEC3G

3
APOBEC3G
  • A 46 kDa innate antiviral factor that is active
    against HIV, SIV, MLV, HTLV-I and likely many
    other retroviruses
  • A cytidine deaminase that belongs to a large
    family of RNA editing/DNA mutator enzymes
    including APOBEC1 and AID
  • Virion incorporation has been proposed to be a
    prerequisite for its antiviral activity

4
HIV Provirus
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In view of the potent anti-viral properties of
APOBEC3G, why are we witnessinga global HIV
epidemic?
  • Because the Vif protein of HIV neutralizes
    APOBEC3G

9
HIV-1 Vif
How does Vif counterthe anti-viral effects of
APOBEC3G?
10
Kim Stopak, JDBMS Graduate Student
11
Stopak et al, Molec Cell, 2003
12
Yu et al, Science, 2003 Marin et al, Nat Med
2003 Sheehy et al, Nat Med 2003 Mehle et al, J.
Biol. Chem 2003
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15
Non-infectious ?Vif HIV
16
Is APOBEC3G subject tosome form of negative
regulationwithin cells?
17
Ya-Lin Chiu, PhD
Vanessa Soros, PhD
18
Endogenous APOBEC3G Is Assembled into a
High-Molecular-Mass (HMM), RNase-sensitive
Complex in H9 Cells
HMM
5-15 Megadaltons
LMM
Western blotting a-APOBEC3G
19
Endogenous APOBEC3G Is Assembled into a
High-Molecular-Mass (HMM), RNase-sensitive
Complex in H9 Cells
HMM
LMM
Western blotting a-APOBEC3G
20
APOBEC3G Enzymatic ActivityAppears to Be
Negatively Regulatedwithin Cells by Recruitment
into a HMM Ribonucleoprotein Complex
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A 20-year Riddle in HIV Biology
  • Why is HIV completely unable to infect resting
    T-cells?
  • APOBEC3G turns out to be a major player

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Knocking-down LMM APOBEC3G Expression in Resting
CD4 T-cells with siRNA
26
siRNA Knockdown of Endogenous APOBEC3G Renders
Unstimulated CD4 T Cells from the Peripheral
Blood Permissive for HIV Infection
27
siRNA Knockdown of Endogenous APOBEC3G Renders
Unstimulated CD4 T Cells from the Peripheral
Blood Permissive for HIV Infection
28
siRNA Knockdown of Endogenous APOBEC3G Renders
Unstimulated CD4 T Cells from the Peripheral
Blood Permissive for HIV Infection
29
LMM APOBEC3G Restricts HIV-1 After Entry into
Resting CD4 T Cells
LMM Complex
Not countered by Vif
Chiu et al, Nature, 2005
30
Recruitment of APOBEC3G into HMM Complexes
Correlates with a Sharp Increase in
Permissiveness for HIV Infection
Activated CD4 T Cells Macrophages H9 T Cell Lines
HMM Complex
Chiu et al, Nature, 2005
31
Purification of HMM NTAP-APOBEC3G Complexfor
Tandem Mass Spectrometry Analysis
NTAP-A3G
32
Tandem Mass SpectrometryResults 95 Different
Proteinsin the HMM APOBEC3G Complex
33
Proteins Identified in HMM APOBEC3G RNP Complexes
34
Does this ensemble of proteins correspond to any
previously known complexes ?
  • Yes
  • Staufen-containing RNA Granules

35
Staufen-containing RNA Granules
  • These gt10 megadalton complexes play a key role
    in localization and regulated translation of
    mRNAs
  • Best studied in Drosophila embryos and mouse
    neurons

36
What RNAs are present in the HMM APOBEC3G
complex?
  • Two retrotransposon (mobile genetic element) RNAs
    detected

37
Retrotransposons and the APOBECs
  • Retrotransposon activity is 100-fold less in
    humans compared to mice
  • Mice contain a single APOBEC3 gene
  • Humans and primates contain 8 APOBEC3 genes
    (tandem duplication)
  • The APOBEC3 genes have undergone striking
    evolution (non-synonomous mutation) suggesting a
    strong selective pressure
  • These changes were evident prior to the
    emergence of the primate lentiviruses could the
    APOBECs be involved in limiting retrotransposon
    activity?

38
Transposable (Mobile) Elements
39
Actions of APOBEC3 Proteins Beyond Blocking
Exogenous Viruses
  • Human A3A, A3B, A3G, and A3F and murine A3
    inhibit murine IAP and MusD
  • Human A3A, A3B and A3F inhibit autonomous non-LTR
    LINE-1 elements
  • Of special interest, retrotransposition of LINE-1
    is not affected by A3G
  • The potential effects of A3G on non-autonomous
    SINEs, including Alu, are unknown

Esnault et al, Nature, 2005 Bogerd et al, NAR,
2006 Chen et al, Curr Biol, 2006 Stenglein
Harris, J Biol Chem, 2006 Turelli et al, J Biol
Chem, 2004
40
Alu
  • One of the most successful human
    retrotransposons
  • Forms 10.5 of the human genome
  • New Alu retrotransposition event occurs in 1 in
    every 100 live births, but decreasing in
    frequency from peak 4050 million years ago
  • Can mediate both insertional and
    post-insertional mutagenic effects

41
The First Type of RNA Identified Alu RNAs
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The Second Type of RNA Identified Human Small Y
RNAs
44
hY RNAs A Novel Class ofL1-dependent,
Non-autonomous, Retrotranspoable Elements
45
APOBEC3G-dependent Recruitment ofAlu and hY RNAs
into Staufen-containing RNA-transporting Granules
46
APOBEC3G-dependent Recruitment ofAlu and hY RNAs
into Staufen-containing RNA-transporting Granules
47
Alu and hY1 RNAs Are Enriched 20-100-fold in HMM
APOBEC3G Complexes
48
mRNAs Encoding Tubulin, Actin, Hsp70, Ro and
Staufen Are Not Detectable in the HMM A3G
Complexes
49
Activation-induced Expression of Alu and hY RNAs
in Peripheral Blood-derived CD4 T-cells
50
LINE-mediated Alu Retrotransposition Assay
51
APOBEC3G Restricts LINE-mediated Alu
Retrotransposition
52
Catalytically Inactive Mutants of A3G Continue to
Function as Effective Inhibitors of Alu
Retrotransposition
53
APOBEC3G-dependent Recruitment of Marked
Alu neoTet Transcripts into Staufen-containing
RNA-transporting Granules in HeLa Cells
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Cell-Virus Interactions as Targets for Drug
Development The Vif-APOBEC3G Axis
Warner C. Greene MD, PhD Director and Senior
Investigator
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High-throughput Screen
  • Objective To find an inhibitor of Vif-mediated
    degradation of APOBEC3G.
  • Approach Screen a library of small molecules
    using a cell line stably expressing
    APOBEC3G-luciferase and conditionally expressing
    Vif (Tet-On Codon Optimized Vif, 7-fold
    stimulation ratio Z-value 0.79).
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