Warner C. Greene MD, PhD Director and Senior Investigator Cell-Virus Interactions as Targets for Drug Development: The Vif-APOBEC3G Axis

Each of us in this room produces a potent innate antiviral factor that can potentially stop HIV “dead in its tracks” 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

HIV Provirus

In view of the potent anti-viral properties of APOBEC3G, why are we witnessing a global HIV epidemic? Because the Vif protein of HIV neutralizes APOBEC3G

HIV-1 Vif How does Vif counter the anti-viral effects of APOBEC3G?

Kim Stopak, JD BMS Graduate Student

Stopak et al, Molec Cell, 2003

Yu et al, Science, 2003 Marin et al, Nat Med 2003 Sheehy et al, Nat Med 2003 Mehle et al, J. Biol. Chem 2003

Why Doesn’t Cellular APOBEC3G Inhibit Wildtype HIV-1 Infection? Noninfectious Producer CellTarget Cell Infectious WT HIV-1 Non-infectious  Vif HIV

Is APOBEC3G subject to some form of negative regulation within cells?

Ya-Lin Chiu, PhD Vanessa Soros, PhD

LMM HMM Western blotting:  -APOBEC3G 5-15 Megadaltons Endogenous APOBEC3G Is Assembled into a High-Molecular-Mass (HMM), RNase-sensitive Complex in H9 Cells

LMM HMM Western blotting:  -APOBEC3G Endogenous APOBEC3G Is Assembled into a High-Molecular-Mass (HMM), RNase-sensitive Complex in H9 Cells

APOBEC3G Enzymatic Activity Appears to Be Negatively Regulated within Cells by Recruitment into a HMM Ribonucleoprotein Complex Active Inactive RNase

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

Knocking-down LMM APOBEC3G Expression in Resting CD4 T-cells with siRNA

siRNA Knockdown of Endogenous APOBEC3G Renders Unstimulated CD4 T Cells from the Peripheral Blood Permissive for HIV Infection HSA (Mo CD24)

siRNA Knockdown of Endogenous APOBEC3G Renders Unstimulated CD4 T Cells from the Peripheral Blood Permissive for HIV Infection HSA (Mo CD24)

siRNA Knockdown of Endogenous APOBEC3G Renders Unstimulated CD4 T Cells from the Peripheral Blood Permissive for HIV Infection HSA (Mo CD24)

LMM APOBEC3G Restricts HIV-1 After Entry into Resting CD4 T Cells Chiu et al, Nature, 2005 LMM Complex Not countered by Vif

Chiu et al, Nature, 2005 HMM Complex Activated CD4 T Cells Macrophages H9 T Cell Lines Recruitment of APOBEC3G into HMM Complexes Correlates with a Sharp Increase in Permissiveness for HIV Infection

Purification of HMM NTAP-APOBEC3G Complex for Tandem Mass Spectrometry Analysis NTAP-A3G

Tandem Mass Spectrometry Results: ~ 95 Different Proteins in the HMM APOBEC3G Complex

Proteins Identified in HMM APOBEC3G RNP Complexes

Does this ensemble of proteins correspond to any previously known complexes ? Yes… Staufen-containing RNA Granules

These >10 megadalton complexes play a key role in localization and regulated translation of mRNAs Best studied in Drosophila embryos and mouse neurons

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

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?

Transposable (Mobile) Elements

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 Actions of APOBEC3 Proteins Beyond Blocking Exogenous Viruses

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 40–50 million years ago Can mediate both insertional and post-insertional mutagenic effects

The First Type of RNA Identified: Alu RNAs

The Second Type of RNA Identified: Human Small Y RNAs

hY RNAs: A Novel Class of L1-dependent, Non-autonomous, Retrotranspoable Elements

APOBEC3G-dependent Recruitment of Alu and hY RNAs into Staufen-containing RNA-transporting Granules

APOBEC3G-dependent Recruitment of Alu and hY RNAs into Staufen-containing RNA-transporting Granules

Alu and hY1 RNAs Are Enriched fold in HMM APOBEC3G Complexes

mRNAs Encoding Tubulin, Actin, Hsp70, Ro and Staufen Are Not Detectable in the HMM A3G Complexes

Activation-induced Expression of Alu and hY RNAs in Peripheral Blood-derived CD4 T-cells

LINE-mediated Alu Retrotransposition Assay

APOBEC3G Restricts LINE-mediated Alu Retrotransposition

Catalytically Inactive Mutants of A3G Continue to Function as Effective Inhibitors of Alu Retrotransposition

APOBEC3G-dependent Recruitment of Marked Alu neo Tet Transcripts into Staufen-containing RNA-transporting Granules in HeLa Cells

HIV

Warner C. Greene MD, PhD Director and Senior Investigator Cell-Virus Interactions as Targets for Drug Development: The Vif-APOBEC3G Axis

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).