Mapping the Vif–A3G interaction using peptide arrays: A basis for anti-HIV lead peptides

Abstract

Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3G (A3G) is a cytidine deaminase that restricts retroviruses, endogenous retro-elements and DNA viruses. A3G plays a key role in the anti-HIV-1 innate cellular immunity. The HIV-1 Vif protein counteracts A3G mainly by leading A3G towards the proteosomal machinery and by direct inhibition of its enzymatic activity. Both activities involve direct interaction between Vif and A3G. Disrupting the interaction between A3G and Vif may rescue A3G antiviral activity and inhibit HIV-1 propagation. Here, mapping the interaction sites between A3G and Vif by peptide array screening revealed distinct regions in Vif important for A3G binding, including the N-terminal domain (NTD), C-terminal domain (CTD) and residues 83-99. The Vif-binding sites in A3G included 12 different peptides that showed strong binding to either full-length Vif, Vif CTD or both. Sequence similarity was found between Vif-binding peptides from the A3G CTD and NTD. A3G peptides were synthesized and tested for their ability to counteract Vif action. A3G 211-225 inhibited HIV-1 replication in cell culture and impaired Vif dependent A3G degradation. In vivo co-localization of full-length Vif with A3G 211-225 was demonstrated by use of FRET. This peptide has the potential to serve as an anti-HIV-1 lead compound. Our results suggest a complex interaction between Vif and A3G that is mediated by discontinuous binding regions with different affinities.

Reassessing APOBEC3G Inhibition by HIV-1 Vif-Derived Peptides

“…Vif peptide 6024 (H-VKHHMYISGKAKGWF-OH), which spans residues 25–39, showed the strongest inhibition (>10-fold), and overlapping Vif peptides 6043 (H-DLADQLIHLYYFDCF-OH) and 6044 (H-QLIHLYYFDCFSESA-OH), which span residues 101–115 and 105–119, respectively, showed weaker inhibition. These data indicate that two, non-overlapping regions of Vif have A3G inhibition activity, consistent with prior studies [14, 16, 18]. …”

“…A previous screen of Vif peptides identified several with A3G inhibitory activity [14, 16]. We started our studies by attempting to reproduce this screen using the same library (HIV-1 lab strain NL4-3 Vif peptide library Cat#6446), a fluorescence-based DNA deaminase activity assay [19], and A3G expressed in the context of a soluble extract from HEK-293 cells (assay schematic in Figure 2A).…”

“…HIV-1 Vif has also been shown to block the single-stranded (ss)DNA deaminase activity of A3G in cells and in viral particles [13–15]. These observations motivated screening of Vif-derived peptides for A3G inhibitory activity [14, 16]. Two peptides spanning Vif regions 25–39 and 105–119 showed low micromolar inhibitory activities in biochemical assays and even evidence for A3G antagonism in living cells [14–18].…”

Scalable high-density peptide arrays for comprehensive health monitoring

“…Peptide arrays have been used to assay kinase activity and inhibitors 23,31 , map monoclonal antibodies 32 , identify autoantigens 33 , even bind proteins 34 and cells 35 . The synthesis fidelity and peptide density of these arrays make it feasible to tile the entire human proteome or the proteomes of most human pathogens for studies of autoimmunity or vaccine development 23,26,36 .…”

Identifying Protein-protein Interaction Sites Using Peptide Arrays

“…Peptides array can be used to identify the sites that mediate protein-protein interactions. Examples of such proteins studied in our lab using this method are: the interaction between the HIV-1 Vif protein and cellular A3G protein was characterized using a peptide array 24 ; peptide array screening was used to study the interactions of the anti-apoptotic BCL-2 family with the pro-apoptotic ASPP2 protein 25 and to study the binding of tBID, a member of BCL-2 family, with the mitochondrial protein MTCH2…”