The structural basis for HIV-1 Vif antagonism of human APOBEC3G

The APOBEC3 (A3) proteins are host antiviral cellular proteins that hypermutate the viral genome of diverse viral families. In retroviruses, this process requires A3 packaging into viral particles1-4. The lentiviruses encode a protein, Vif, that antagonizes A3 family members by targeting them for degradation. Diversification of A3 allows host escape from Vif whereas adaptations in Vif enable cross-species transmission of primate lentiviruses. How this 'molecular arms race' plays out at the structural level is unknown. Here, we report the cryogenic electron microscopy structure of human APOBEC3G (A3G) bound to HIV-1 Vif, and the hijacked cellular proteins that promote ubiquitin-mediated proteolysis. A small surface explains the molecular arms race, including a cross-species transmission event that led to the birth of HIV-1. Unexpectedly, we find that RNA is a molecular glue for the Vif-A3G interaction, enabling Vif to repress A3G by ubiquitin-dependent and -independent mechanisms. Our results suggest a model in which Vif antagonizes A3G by intercepting it in its most dangerous form for the virus-when bound to RNA and on the pathway to packaging-to prevent viral restriction. By engaging essential surfaces required for restriction, Vif exploits a vulnerability in A3G, suggesting a general mechanism by which RNA binding helps to position key residues necessary for viral antagonism of a host antiviral gene

Screening for AMPA receptor auxiliary subunit specific modulators

<div><p>AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP <i>γ</i>-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC₅₀/EC₅₀s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes.</p></div

Behavior of established compounds in VSD assay.

<p><b>(A1)</b> Normalized fluorescence data for VSD assay on A2R-stg cells with 50 μM fluorowillardiine (black) with maxGLU (red) and NBQX (pink) controls, all normalized to EC₅₀GLU (blue). <b>(A2)</b> CRC curves for FW against A2-stg and (<b>A3)</b> A2-C3 cell lines calculated from the CMPDslope window. %max GLUslope = (CMPDslope—mean VHLslope)/(mean maxGLUslope—mean VHLslope) is further described in methods. (<b>B1)</b> Normalized fluorescence data for VSD assay on A2R-stg cells with 1 mM CX-546 (black) with maxGLU (red) and NBQX (pink) controls, all normalized to EC₅₀GLU (blue). <b>(B2)</b> CRC curves for CX-546 against A2R-stg and (<b>B3)</b> A2R-C3 cell lines calculated from the GLUslope window. %max GLUslope = (GLUslope—mean VHLslope)/(mean maxGLUslope—mean VHLslope) <b>(C1)</b> Normalized fluorescence data for VSD assay on A2R-stg cells with 500 μM cyclothiazide (CTZ, black) with maxGLU (red) and NBQX (pink), all normalized to EC₅₀GLU (blue). <b>(C2)</b> CRC curves for CTZ against A2-stg and <b>(C3)</b> A2-C3 cell lines calculated from the GLUslope window. Compound EC₅₀ values that could be reliably calculated are in the top left corner of each graph.</p

Characterization of VU0627849.

<p><b>(A)</b> Raw data for compound CRCs in the VSD assay. A2R-stg (orange) and A2R-C3 (blue). EC₅₀GLU traces are represented by dashed lines. Concentrations of compound are indicated in the top left corner. <b>(B)</b> CRCs calculated from GLUslope in the VSD assay for A2R-stg and A2R-C3. <b>(C)</b> Raw data for compound CRCs in the calcium flux assay. A2Q (red), A2Q-stg (orange), A2Q-GSG (green), and A2Q-C3 (blue). 1 mM glutamate traces are represented by dashed lines. <b>(D)</b> CRCs calculated from GLUslope1 in calcium flux assay for A2Q-stg, A2Q-C3, A2Q-GSG, and A2Q cell lines. Plotted as %max GLUslope vs. log [compound].</p

Workflow for identifying AMPAR-auxiliary subunit modulators.

<p><b>(A)</b> 39,202 compounds were initially screened using the VSD assay against A2R-stg cells. <b>(B)</b> 1,184 hits from (A) were counter-screened against A2R, TetON, and A2R-C3 cells. <b>(C)</b> 116 compounds were identified from counter-screening in (B) as being stargazin or auxiliary subunit specific (i.e. they did not hit on A2R or TetON cells). These were tested for full compound CRCs against A2R-stg and A2R-C3 cells using the VSD assay. These CRCs identified 90 hits that fit to sigmoidal dose response curves with potency under 10 μM. <b>(D)</b> We identified 39 stargazin specific PAMs, 2 CNIH3 specific PAMs, and 36 PAMs that had activity in both A2R-stg and A2R-C3 cells. We also found 1 stargazin specific NAM and 9 compounds with NAM activity on both cell lines. Three compounds gave opposite effects in the two cell lines. Hits were discarded for reorder if they showed activity in the compound only window. Hits with activity in the CMPD only windows were discarded. <b>(E)</b> 57 of the 90 compounds in (D) were re-screened with new batch samples as compound CRCs in the VSD assay. <b>(F)</b> 57 hits were tested in the glutamate potency fold-shift calcium flux assay and 28 were subjected to a full compound CRC calcium flux assay to study their effects using an orthogonal approach.</p

Chemical structures of our candidate hits.

<p><b>(A)</b> Structure of VU0612951 highlighting the 1,3-triazole group in red. <b>(B)</b> Structure of VU0627849 highlighting the isoxazole group in red. <b>(C)</b> Structure of VU0539491 highlighting the 1,2,4-oxadiazole group in red.</p

Electrophysiology with VU0627849.

<p><b>(A</b>) Whole-cell recordings of A2R cell line with (blue) and without (red) VU0627849 (40μM). <b>(B)</b> Whole-cell recordings of A2R-stg cell line with (blue) and without (red) VU0627849 (40μM). Recording after washout of drug is in black. In these experiments, glutamate (1mM) is applied for 100 ms and 20 ms pulses with or without VU0627849.</p

Characterization of VU0539491.

<p><b>(A)</b> Raw data for compound CRCs in the VSD assay. A2R-stg (orange) and A2R-C3 (blue). EC₅₀GLU traces are represented by dashed lines. Compound concentrations are indicated in the top left corner. <b>(B)</b> CRCs calculated from GLUslope in VSD assay for A2R-stg and A2R-C3. <b>(C)</b> Raw data for compound CRCs in the calcium flux assay. A2Q (red), A2Q-stg (orange), A2Q-GSG (green), and A2Q-C3 (blue). Traces obtained from applying 1 mM glutamate are represented by dashed lines. <b>(D)</b> Compound CRCs calculated from the GLUslope1 window in our calcium flux assay for A2Q-stg, A2Q-C3, A2Q-GSG, and A2Q cell lines. These show negative and positive trends but no curve fits. <b>(E)</b> Compound CRCs calculated from the AUC in the GLUmaxmin window of the calcium flux assay plotted as %max AUC vs. log [compound] as in (7E).</p

Summary of cell lines used for screening.

<p>Summary of cell lines used for screening.</p