Warner C' Greene MD, PhD

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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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Each of us in this roomproduces a potent innate
antiviral factorthat can potentially stop
HIVdead in its tracks

• APOBEC3G

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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

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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

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HIV-1 Vif
How does Vif counterthe anti-viral effects of
APOBEC3G?
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Kim Stopak, JDBMS Graduate Student
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Stopak et al, Molec Cell, 2003
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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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Non-infectious ?Vif HIV
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Is APOBEC3G subject tosome form of negative
regulationwithin cells?
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Ya-Lin Chiu, PhD
Vanessa Soros, PhD
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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
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Endogenous APOBEC3G Is Assembled into a
High-Molecular-Mass (HMM), RNase-sensitive
Complex in H9 Cells
HMM
LMM
Western blotting a-APOBEC3G
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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
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siRNA Knockdown of Endogenous APOBEC3G Renders
Unstimulated CD4 T Cells from the Peripheral
Blood Permissive for HIV Infection
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siRNA Knockdown of Endogenous APOBEC3G Renders
Unstimulated CD4 T Cells from the Peripheral
Blood Permissive for HIV Infection
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siRNA Knockdown of Endogenous APOBEC3G Renders
Unstimulated CD4 T Cells from the Peripheral
Blood Permissive for HIV Infection
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LMM APOBEC3G Restricts HIV-1 After Entry into
Resting CD4 T Cells
LMM Complex
Not countered by Vif
Chiu et al, Nature, 2005
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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
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Purification of HMM NTAP-APOBEC3G Complexfor
Tandem Mass Spectrometry Analysis
NTAP-A3G
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Tandem Mass SpectrometryResults 95 Different
Proteinsin the HMM APOBEC3G Complex
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Proteins Identified in HMM APOBEC3G RNP Complexes
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Does this ensemble of proteins correspond to any
previously known complexes ?

• Yes
• Staufen-containing RNA Granules

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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

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What RNAs are present in the HMM APOBEC3G
complex?

• Two retrotransposon (mobile genetic element) RNAs
  detected

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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?

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Transposable (Mobile) Elements
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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
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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

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The First Type of RNA Identified Alu RNAs
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The Second Type of RNA Identified Human Small Y
RNAs
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hY RNAs A Novel Class ofL1-dependent,
Non-autonomous, Retrotranspoable Elements
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APOBEC3G-dependent Recruitment ofAlu and hY RNAs
into Staufen-containing RNA-transporting Granules
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APOBEC3G-dependent Recruitment ofAlu and hY RNAs
into Staufen-containing RNA-transporting Granules
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Alu and hY1 RNAs Are Enriched 20-100-fold in HMM
APOBEC3G Complexes
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mRNAs Encoding Tubulin, Actin, Hsp70, Ro and
Staufen Are Not Detectable in the HMM A3G
Complexes
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Activation-induced Expression of Alu and hY RNAs
in Peripheral Blood-derived CD4 T-cells
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LINE-mediated Alu Retrotransposition Assay
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APOBEC3G Restricts LINE-mediated Alu
Retrotransposition
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Catalytically Inactive Mutants of A3G Continue to
Function as Effective Inhibitors of Alu
Retrotransposition
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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).