Zinc binding to the HCCH motif of HIV-1 virion infectivity factor induces a conformational change that mediates protein–protein interactions

Abstract

Virion infectivity factor (Vif) is an accessory protein encoded by HIV-1 and is critical for viral infection of the host CD4 ؉ T cell population. Vif induces ubiquitination and subsequent degradation of Apo3G, a cytosolic cytidine deaminase that otherwise targets the retroviral genome. Interaction of Vif with the cellular Cullin5-based E3 ubiquitin ligase requires a conserved BC box and upstream residues that are part of the conserved H-(Xaa)5-C-(Xaa)17-18-C-(Xaa)3-5-H (HCCH) motif. The HCCH motif is involved in stabilizing the Vif-Cullin 5 interaction, but the exact role of the conserved His and Cys residues remains elusive. In this report, we find that full-length HIV-1 Vif, as well as a HCCH peptide, is capable of binding to zinc with high specificity. Zinc binding induces a conformational change that leads to the formation of large protein aggregates. EDTA reversed aggregation and regenerated the apoprotein conformation. Cysteine modification studies with the HCCH peptide suggest that C114 is critical for stabilizing the fold of the apopeptide, and that C133 is located in a solvent-exposed region with no definite secondary structure. Selective alkylation of C133 reduced metal-binding specificity of the HCCH peptide, allowing cobalt to bind with rates comparable to that with zinc. This study demonstrates that the HCCH motif of HIV-1 Vif is a unique metal-binding domain capable of mediating protein-protein interactions in the presence of zinc and adds to a growing list of examples in which metal ion binding induces protein misfolding and/or aggregation.aggregation ͉ cullin ubiquitin ligase ͉ metal-binding protein

Core Binding Factor Beta Plays a Critical Role by Facilitating the Assembly of the Vif-Cullin 5 E3 Ubiquitin Ligase

“…Vif binds EloC through its BC box region (residues 144 SLQYLA 149 ) (13)(14)(15)(16), which mimics the conserved cellular interface of SOCS box proteins. The interaction between Vif and Cul5 is primarily mediated by a novel zinc-binding motif (H 108 C 114 C 133 H 139 ) upstream of the Vif BC box (17)(18)(19)(20)(21).…”

Core-Binding Factor Subunit Beta Is Not Required for Non-Primate Lentiviral Vif-Mediated APOBEC3 Degradation

“…HIV-1 viral infectivity factor (Vif) is essential for viral infectivity because it recruits the host restriction factor APOBEC3G (A3G) to the E3 ligase complex, which consists of the scaffold protein Cullin5 (CUL5) and substrate adaptors Elongin B/C and Rbx, to induce A3G polyubiquitination and degradation, thereby suppressing A3G-mediated antiviral activity (6,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Recent studies have shown that the transcription cofactor core-binding factor subunit beta (CBF-␤) is a key factor in Vif function (29,30).…”

Regulation of APOBEC3 Proteins by a Novel YXXL Motif in Human Immunodeficiency Virus Type 1 Vif and Simian Immunodeficiency Virus SIVagm Vif

“…Vif may also inhibit APOBEC3 activity through mechanisms independent of proteasomal degradation (17,18,27,36,46). Vif associates with the Cul5-EloB-EloC complex by binding directly to EloC via a BC box motif at positions 144 to 153 and to Cul5 via hydrophobic residues at positions 120, 123, and 124 within a zincbinding region (residues 100 to 142) formed by a conserved H-X 5 -C-X 17-18 C-X 3-5 -H (HCCH) motif and a recently identified Vif cullin box (26,28,33,45). Vif binding to A3G and A3F is essential for their degradation by the Vif-Cul5 E3 ligase (25,27,29).…”