Identification of Specific Determinants of Human APOBEC3F, APOBEC3C, and APOBEC3DE and African Green Monkey APOBEC3F That Interact with HIV-1 Vif

Abstract

Human APOBEC3F (hA3F) and human APOBEC3G (hA3G) are potent anti-human immunodeficiency virus (anti-HIV) host factors that suppress viral replication by hypermutating the viral genome, inhibiting reverse transcription, and hindering integration. To overcome hA3F and hA3G, HIV-1 encodes Vif, which binds and targets these host proteins for proteasomal degradation. Previously, we reported that the hA3F-Vif interactions that lead to hA3F degradation are located in the region comprising amino acids 283 to 300. We have now performed mutational analysis of this region and found that the 289 EFLARH 294 amino acids contribute to hA3F-Vif binding and are critical for A3F's sensitivity to Vif. Mutants in which E289 is mutated significantly increase hA3F's ability to inhibit viral infectivity in the presence of Vif, and coimmunoprecipitation assays show that binding of Vif to the E289K mutant is decreased. We examined the role of the EFLARH sequence in other A3 proteins, including human A3C (hA3C), human A3DE (hA3DE), African green monkey A3F (agmA3F), and rhesus macaque A3F (rhA3F). hA3C, hA3DE, and agmA3F were all susceptible to degradation induced by HIV-1 Vif, while rhA3F was not. Mutagenesis of the glutamate in the EFLARH sites of hA3C, hA3DE, and agmA3F decreases the susceptibilities of these proteins to Vif-induced degradation. Together, these results indicate that the EFLARH region in hA3F, hA3C, hA3DE, and agmA3F interacts with HIV-1 Vif and that this interaction plays a role in the Vif-mediated proteasomal degradation of these A3 proteins. These studies identify a conserved region in 3 of 7 human A3 proteins that is critical for degradation mediated by HIV-1 Vif and provide structural insights into the hA3F-Vif interactions that could facilitate the development of a novel class of anti-HIV agents.

HIV-1 and HIV-2 Vif Interact with Human APOBEC3 Proteins Using Completely Different Determinants

“…Previously, we and others found that the determinant in A3G for Vif1-induced degradation includes D128 and the surrounding residues (45, 55-57), and we found that the critical determinant in A3F for Vif1-induced degradation includes E289 and the surrounding residues (42,58). A3G D128K and GFG2 mutants (9) and A3F E289K and FGF4 mutants (7), were previously shown to be resistant to Vif1-induced degradation (Fig.…”

“…6, the A3FϾ5A mutant, in which A3F box 1 amino acids were replaced by alanines, was sensitive to Vif2. In addition, a chimera that we previously reported (42), in which residues 125 to 131 in box 1 of A3F were replaced by the corresponding residues of A3G, was Vif2 sensitive (Fig. 8B).…”

“…The pFLAG-A3C, -A3D, -A3F, -A3G, -A3H-hapII, -A3H-hapI, -A3G-D128K, -A3F-E289K, -GFG2, and -FGF4 plasmids were previously described (34,(40)(41)(42). Myc-A3A-HA was kindly provided by Gisela Heidecker and David Derse (National Cancer Institute-Frederick).…”

“…We previously used alanine-scanning mutagenesis to identify two distinct regions in the first 60 amino acids of Vif1 that are critical for neutralization of A3F and A3G (34,40,42). An alignment of the N termini of Vif1 and Vif2 shows that these proteins are dissimilar ( Fig.…”

APOBEC3F Determinants of HIV-1 Vif Sensitivity

“…A3F provides a unique opportunity to advance this goal because the crystal structure of its catalytic domain, which also binds Vif, has been solved (9,10). Previous studies have interrogated this interaction by manipulating human A3F (huA3F), which is normally sensitive to HIV-1 Vif-mediated degradation, to become resistant to Vif-mediated degradation (11)(12)(13). Here, the reciprocal approach is employed to determine the regions of A3F that mediate its sensitivity to HIV-1 Vif.…”