Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G

<div><p>Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.</p> </div

UBE2F is required for activation of CRL5^Vif-CBFß<i>in vitro</i>.

<p><b>A,</b> Diagram of the ubiquitination protocol used in panel <b>B</b>. <b>B, </b><i>In vitro</i> ubiquitination of A3G by recombinant CRL5^Vif-CBFß with UBE2R1 as ubiquitin conjugating enzyme requires UBE2F. Immunoblots of ubiquitination reactions containing myc-tagged A3G as the substrate show high-molecular weight polyubiquitin chains, require all protein components of the ubiquitin and NEDD8 activating systems and are only observed when UBE2F (lane 9) but not when UBE2M (lane 10) is used as NEDD8 conjugating enzyme. A3A is not susceptible to Vif and was used as a negative control. <b>C,</b> Coomassie-stained SDS-PAGE of NEDD8ylation “pulse” reaction indicates that under conditions used in panel <b>B</b> indicate CUL5 is completely NEDD8ylated by UBE2F; only a minor fraction (<5%) is NEDD8ylated by UBE2M. <b>D, </b>¹⁵N-HSQC spectral overlays of RBX2_RING in the presence and absence of ∼100 µM, unlabeled full-length UBE2M (top) or UBE2F (bottom).</p

A model illustrating how inhibition of CUL5 NEDD8ylation leads to reduced infectivity of HIV.

<p>Two enzymatic steps must take place in order for CRL5^Vif-CBFß to be properly activated by NEDD8 conjugation, and therefore for A3G-degradation to take place, in cells infected with HIV. First, NEDD8 is loaded onto the E2 UBE2F by NAE. The small molecule MLN4924 is able to inhibit this step, blocking degradation of A3G and thereby reducing viral infectivity. Second, UBE2F is recognized by the RBX2 subunit of CRL5^Vif-CBFß, and transfers NEDD8 to CUL5.</p

The UBE2F loop insertion is required for efficient viral infectivity.

<p><b>A,</b> Single-cycle infectivity assay of HIV_NL4-3ΔVif produced from HEK293T cells stably depleted for UBE2F (white bars), or non-silencing control shRNA (black bars) in the presence of transfected HIV_NL4-3ΔVif (1 µg), A3G-V5 (500 ng), Vif-FLAG (100 ng), and increasing amounts of RNAi-immune wild-type or ΔLoop UBE2F-myc (0.2, 1, 3 or 10 ng), a catalytic mutant of UBE2F harboring a cysteine to alanine change at position 116 (10 ng), or empty vector control (10 ng). Mean and +-SD of duplicate experiments are graphed. <b>B,</b> Immunoblots of cell lysates and virus particles corresponding to panel <b>A</b>. A3G levels in cellular lysates and virus particles in UBE2F KD cells transfected with increasing amounts of RNAi-immune UBE2F (compare lanes 3–6, left and right), catalytic mutant UBE2F (C116A) or increasing amounts of UBE2F (ΔLoop) (lanes 1, 2, 7–10 left and right). To discern A3G levels in the virion in the shSCR lanes the immnoblots were exposed longer and non-specific bands from the protein ladder became apparent, as indicated by an asterisk.</p

Pharmacological inhibition of NEDD8 E1 by MLN4924 blocks the ability of Vif to counteract A3G.

<p><b>A,</b> Single-cycle infectivity assay of HIV_NL4-3 produced in HEK293T cells transfected with empty vector control (white bars) or V5-tagged A3G (black bars, 120 ng) , 1 µg of NL4-3 proviral DNA and treated with indicated concentrations of MLN4924. <b>B,</b> Parallel immunoblots indicating MLN4924 restores steady-state levels of A3G in cells and packaging in virions. <b>C,</b> Quantitation of G to A mutations in gDNA sequences from virions produced in cells treated with either DMSO or 500 nM MLN4924.</p

Structural basis for discrimination between NEDD8 pathways.

<p><b>A,</b> Surface representations of RBX1_RING and RBX2_RING with divergent surface residues (Swap4) targeted for substitution analysis. Coordinates for RBX1, RBX2 were from PDB files 2EDI and 3DQV, respectively. <b>B,</b> Time courses of NEDD8 transfer from UBE2M to indicated CUL5/RBX complexes and <b>C,</b> relative initial rates for NEDD8ylation normalized to CUL5/RBX1. Error bars indicate standard deviation between at least two experiments. <b>D,</b> Excerpts from ¹⁵N-HSQC spectra of RBX2 titrated with increasing concentrations of unlabelled wild-type or mutant UBE2F. <b>E,</b> Time courses for NEDD8 transfer from wild-type and UBE2F mutants to CUL5/RBX2. Error bars indicate standard deviation between at least two experiments. <b>F,</b> Relative initial rates for NEDD8ylation of CUL5/RBX2 normalized by wild-type UBE2F. Error bars indicate standard deviation between at least two experiments. <b>G,</b> Model of CUL5/RBX2/UBE2F complex based on CUL1/RBX1 crystal structure (3RTR) and cIAP2/UBE2D2 crystal structure (3EB6) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003085#ppat.1003085-Calabrese1" target="_blank">[33]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003085#ppat.1003085-Mace1" target="_blank">[60]</a>. UBE2F, RBX2 and CUL5 are shown in white, grey, and green respectively. The catalytic cysteine of UBE2F and the NEDD8 acceptor lysine of CUL5 (K724) are shown in orange; the unique loop insertion of UBE2F, blue; Asn92 of UBE2F, yellow sticks; and residues of RBX2 targeted for Swap4 mutation, red. Details of modeling can be found in the Experimental Procedures.</p