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Background:  APOBEC3G, a newly identified antiretroviral host factor, is a cytidine deaminase that induces G-to-A hypermutation in the newly synthesized viral DNA to inhibit HIV-1 replication. Several groups have shown that HIV-1 Vif binds to E3 ubiquitin ligase complex composed of Cullin5 (Cul5), Elongin B (Elo B), and Elongin C (Elo C) through its SOCS-box-like motif and that their dominant negative mutants or proteasome inhibitors suppress the degradation of APOBEC3G by HIV-1 Vif. This indicates that HIV-1 Vif overcomes APOBEC3G by the ubiqutin-proteasome pathway. However, there has been no direct evidence on the ubiquitination of APOBEC3G by a Vif-Cul5-Elo B/C complex. In this study, we examined the ubiquitination of APOBEC3G by in vitro ubiquitin conjugation assay using baculovirus-produced proteins.

Methods:  We infected HiFive cells with recombinant baculoviruses expressing His-vif, Cul5, Elo B, Elo C, and Rbx1. Purification of His-Vif protein with NTA beads showed the formation of a Vif-Cul5-Elo B/C complex. We purified HA-APOBEC3G and HA-D128K as substrates by immunoprecipitation with an anti-HA monoclonal antibody from 293T cells transfected with expression vectors. In vitro ubiquitin conjugation assay was performed by mixing a Vif-Cul5-Elo B/C complex, substrates, and several purified proteins necessary for this reaction such as E1, E2, Nedd8, APP-BP, Ubc12, and GST-Ub. After incubation, samples were subjected to immunoblotting with an anti-HA monoclonal antibodies.

Results:  This assay clearly showed the polyubiquitination of APOBEC3G by a Vif-Cul5-Elo B/C complex, but not that of D128K which could not bind to HIV-1 Vif.

Conclusions:  HIV-1 Vif could form a Vif-Cul5-Elo B/C complex and this complex acts as E3 ligase to conjugate ubiquitins to APOBEC3G. This also suggests that this complex is sufficient for the ubiquitination of APOBEC3G in vitro.

Keywords: HIV-1 Vif; APOBEC3G; Ubiquitination