Figure 1.
Diversifying selection of the Vif binding site of APOBEC3G in the Cercopithecinae subfamily of Old World Monkeys.
A partial primate species phylogeny [37] is depicted as a cladogram and accompanied by partial amino acid sequences of respective A3G orthologs. Approximate divergence times (in millions of years) are placed at relevant ancestral nodes. Select residues comprising the putative site targeted by Vif proteins are shown. Primates of the order Catarrhini, consisting of hominoids (blue) and Old World Monkeys, are included. Old World Monkeys are subdivided into the Cercopithecinae (green) and Colobinae (red) subfamilies. Sources of sequences previously reported elsewhere are indicated by reference number. # = new sequences reported in this study, n = number of individuals analyzed per species.
Figure 2.
Adaptive evolution of A3G at codons 128 and 130 allows escape from Vif-mediated antagonism.
Single-round infectivity assays were performed with HIV-1ΔVif and HIV-1 expressing SIV Vif proteins produced in the presence of A3G variants from monkeys of the Cercopithecus genus in (A) and (C). Infectivity of viruses is reported as a percentage, relative to infectivity in the absence of A3G (100%). Error bars indicate standard deviation from the mean of two independent transfection experiments (six infection replicates in total). The Vif binding site of each A3G variant is depicted in grey boxes. (A) and (C) The primate A3G gene used is listed under the graph, while the source of the Vif genes were either no Vif (black box), SIVagm.ver (orange), SIVagm.sab (yellow), SIVdeb (blue), SIVagm.gri (green), or SIVmus-1 (golden). Anti-HA western blot analysis was used to measure A3G expression in virus producer cells in (B) and (D). Anti-ß-actin served as protein loading controls.
Figure 3.
Broad and potent activity of Vif from SIVsm and its descendants allow antagonism of rhesus and human A3G variants.
Single-round infectivity assays were performed with HIV-1ΔVif and HIV-1 expressing SIV Vif proteins produced in the presence of A3G variants from human and rhesus macaques (A). Infectivity of viruses is reported as a percentage, relative to infectivity in the absence of A3G (100%). Error bars indicate standard deviation from the mean of two independent transfection experiments (six infection replicates in total). The Vif binding site of each A3G variant is depicted in grey boxes. The primate A3G gene used is listed under the graph, while the source of the Vif genes were either no Vif (black box), SIVagm.ver (orange), HIV-1 (grey), SIVsm (light blue), SIVmac (dark purple), or HIV-2 (light purple). Anti-HA western blot analysis was used to measure human A3G expression in (B) and rhesus A3G expression in (C). Anti-ß-actin served as protein loading controls.
Table 1.
Sensitivity of A3G variants to a spectrum of diverse SIV Vif proteins.
Figure 4.
A multi-residue insertion that blocks Vif emerged 12 MYA in A3G of the Colobinae ancestor.
(A) Single-round infectivity assays were performed with HIV-1ΔVif and HIV-1 expressing SIV Vif proteins produced in the presence of colobus/AGM chimeric A3G proteins. Infectivity of viruses is reported as a percentage, relative to infectivity in the absence of A3G (100%). Error bars indicate standard deviation from the mean of two independent transfection experiments (six infection replicates in total). (B) Partial protein alignment of A3G orthologs reveals a three-residue insertion (red) unique to members of the Colobinae subfamily of OWM. Members of primate parvorders Catarrhini (Old World Monkeys and Hominoids) and Platyrrhini (New World Monkeys) are included. Old World Monkeys are further divided into subfamilies Cercopithecinae and Colobinae. (C) Residues of A3G responsible for differential sensitivity to SIVsab and SIVolc Vif. The canonical Vif binding site is boxed in yellow. Divergent character states independently selected at residues 128 and 130 (underlined) of Cercopithecinae A3G are displayed below. The three-residue insertion in the N-terminus of colobus A3G is bolded in red. The residues of A3G required for antagonism by SIVolc Vif, as found in this study, are underlined and boxed in green. Divergent character states at residues 133, 137 and 145 identified in Colobinae A3G are displayed below. (D) and (E) Single-round infectivity assays were performed with HIV-1ΔVif and HIV-1 expressing SIVolc Vif produced in the presence of mutated colobus/AGM chimeras.