Background:  HIV-1 Vif inactivates a host cell antiviral defense mechanism mediated by the cytidine deaminase, APOBEC3G. In the absence of normal Vif function, APOBEC3G is packaged into HIV-1 virions, causing hypermutation of the viral DNA during reverse transcription in the next round of infection. Vif prevents this by binding directly to APOBEC3G and inducing its degradation. Sites of interaction between Vif and APOBEC3G have not been precisely defined. To better define regions involved in the Vif-APOBEC3G interaction, we used an in vitro screening assay to identify peptides derived from either HIV-1 Vif or human APOBEC3G that inhibit this interaction.

Methods:  An in vitro Vif-APOBEC3G binding assay was used to screen for peptides, derived either from an HIV-1 Vif peptide library or from the N-terminal region of human APOBEC3G, which could block this protein-protein interaction. GST-Vif bound to glutathione-agarose beads was incubated with human APOBEC3G and individual peptides. Following incubation, the beads were collected and the level of APOBEC3G binding to Vif was determined by SDS-PAGE and Western blotting using an anti-human APOBEC3G monoclonal antibody.

Results:  We identified peptides from 2 regions of Vif, separated by 30 amino acid residues, which specifically inhibited the interaction between APOBEC3G and GST-Vif in vitro. One of these peptides had an estimated IC₅₀ of 200 nM and formed a stable complex with APOBEC3G, supporting the hypothesized mechanism of inhibition. Initial screening of several APOBEC3G peptides identified 1 peptide that inhibited APOBEC3G binding to GST-Vif in vitro with an estimated IC₅₀ of 1 mM. In addition this peptide specifically blocked the direct binding of APOBEC3G to the aforementioned inhibitory Vif peptide.

Conclusions:  We have identified Vif and APOBEC3G peptides that specifically inhibit the interaction between human APOBEC3G and HIV-1 Vif in vitro. The identified peptides provide information about the amino acid sequences that may be involved in the interaction of these 2 proteins. This information will facilitate structure/function studies that may lead to the development of rationally designed antiviral agents that target this important intracellular protein–protein interaction in HIV-1-infected cells.