Analysis of Vif-induced APOBEC3G degradation using an alpha-complementation assay.
Vif forms a complex with Elongin B/C, Cullin-5 and Rbx-1 to induce the polyubiquitination and proteasome-mediated degradation of human APOBEC3G (hA3G). These interactions serve as potential targets for anti-HIV-1 drug development. We have developed a cell culture-based assay to measure Vif-induced hA3G degradation. The assay is based on alpha-complementation, the ability of beta-galactosidase fragments to complement in trans. hA3G expressed with a fused alpha-peptide was enzymatically active, complemented a coexpressed omega-fragment and could be targeted for degradation by Vif. Vif reduced beta-galactosidase activity in the cell by 10-30-fold. The assay was validated by testing various hA3G and Vif point mutants. The assay accurately detected the effects of D128 in hA3G, and the BC box, Cul5 box and HCCH motifs of Vif. The results showed a strict association of Vif biological function with hA3G degradation. These findings support hA3G degradation as a requirement for Vif function. The Vif alpha-complementation assay may be a useful tool for the identification of Vif inhibitors.
Fang
Lei
L
Infectious Disease Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037-1099, USA.
Landau
Nathaniel R
NR
D000071480:APOBEC-3G Deaminase; D020816:Amino Acid Motifs; D002460:Cell Line; D003564:Cytidine Deaminase; D016346:Gene Products, vif / Q000502:physiology*; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006801:Humans; D009700:Nucleoside Deaminases / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D014773:Virology / Q000379:methods*; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
D016346:Gene Products, vif; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase; D009700:Nucleoside Deaminases; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase