Abstract
Human immunodeficiency virus-1 (HIV-1) Vif overcomes the anti-viral activity of APOBEC3G by targeting it for ubiquitination via a Cullin 5-ElonginB-ElonginC (Cul5-EloBC) E3 ligase. Vif associates with Cul5-EloBC through a BC-box motif that binds EloC, but the mechanism by which Vif selectively recruits Cul5 is poorly understood. Here we report that a region of Vif (residues 100-142) upstream of the BC-box binds selectively to Cul5 in the absence of EloC. This region contains a zinc coordination site HX5CX17-18CX3-5H (HCCH), with His/Cys residues at positions 108, 114, 133, and 139 coordinating one zinc ion. The HCCH zinc coordination site, which is conserved among primate lentivirus Vif proteins, does not correspond to any known class of zinc-binding motif. Mutations of His/Cys residues in the HCCH motif impair zinc coordination, Cul5 binding, and APOBEC3G degradation. Mutations of conserved hydrophobic residues (Ile-120, Ala-123, and Leu-124) located between the two Cys residues in the HCCH motif disrupt binding of the zinc-coordinating region to Cul5 and inhibit APOBEC3G degradation. The Vif binding site maps to the first cullin repeat in the N terminus of Cul5. These data suggest that the zinc-binding region in Vif is a novel cullin interaction domain that mediates selective binding to Cul5. We propose that the HCCH zinc-binding motif facilitates Vif-Cul5 binding by playing a structural role in positioning hydrophobic residues for direct contact with Cul5.
Highlights
Ubiquitination requires the coordinated activity of an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and a substrate-specific E3 ubiquitin ligase [24]
The Elongins B and C (EloBC) dimer serves as the adaptor molecule that bridges BC-box proteins to the cullin scaffold by binding the N terminus of the cullin and the BC-box, an ϳ10-amino-acid degenerate sequence motif ((Ala/Pro/Ser/Thr)-Leu-Xaa3-(Cys/Ala)-Xaa3-(Ala/Ile/ Leu/Val))
The conserved cysteines at positions 114 and 133 upstream of the BC-box (Fig. 1A) are required for Cullin 5 (Cul5) association [19, 20, 27], APOBEC3G degradation, and viral replication [15, 16, 32]. These cysteines are part of a highly conserved HX5CX17–18CX3–5H (HCCH) motif present in all primate lentivirus Vif proteins (Fig. 1A and supplemental Fig. 1). This conserved HCCH motif was shown to be required for assembly of the Vif-Cul5 E3 ligase that ubiquitinates APOBEC3G [19, 20, 27]
Summary
Antibodies and Plasmids—Antibodies used were rabbit anti-Vif [28], 3F10 anti-HA (Roche Applied Science), anti-T7 (Novagen), 9B11 antiMyc (Cell Signal), M2 anti-FLAG (Sigma), rabbit anti-Cul
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