Identification of an APOBEC3G binding site in human immunodeficiency virus type 1 Vif and inhibitors of Vif-APOBEC3G binding.
The APOBEC3 cytidine deaminases are potent antiviral factors that restrict replication of human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif binds APOBEC3G and APOBEC3F and targets these proteins for ubiquitination by forming an E3 ubiquitin ligase with cullin 5 and elongins B and C. The N-terminal region of Vif is required for APOBEC3G binding, but the binding site(s) is unknown. To identify the APOBEC3G binding site in Vif, we established a scalable binding assay in a format compatible with development of high-throughput screens. In vitro binding assays using recombinant proteins identified Vif peptides and monoclonal antibodies that inhibit Vif-APOBEC3G binding and suggested involvement of Vif residues 33 to 83 in APOBEC3G binding. Cell-based binding assays confirmed these results and demonstrated that residues 40 to 71 in the N terminus of Vif contain a nonlinear binding site for APOBEC3G. Mutation of the highly conserved residues His42/43 but not other charged residues in this region inhibited Vif-APOBEC3G binding, Vif-mediated degradation of APOBEC3G, and viral infectivity. In contrast, mutation of these residues had no significant effect on Vif binding and degradation of APOBEC3F, suggesting a differential requirement for His42/43 in Vif binding to APOBEC3G and APOBEC3F. These results identify a nonlinear APOBEC3 binding site in the N terminus of Vif and demonstrate that peptides or antibodies directed against this region can inhibit Vif-APOBEC3G binding, validating the Vif-APOBEC3 interface as a potential drug target.
Mehle
Andrew
A
Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Wilson
Heather
H
Zhang
Chengsheng
C
Brazier
Andrew Jay
AJ
McPike
Mark
M
Pery
Erez
E
Gabuzda
Dana
D
D000071480:APOBEC-3G Deaminase; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D003564:Cytidine Deaminase / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
D054320:vif Gene Products, Human Immunodeficiency Virus; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase