Structure-Guided Analysis of the Human APOBEC3-HIV Restrictome Halil Aydin, Matthew W. Taylor, Jeffrey E. Lee  Structure  Volume 22, Issue 5, Pages 668-684 (May 2014) DOI: 10.1016/j.str.2014.02.011 Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 1 Antiviral Activities of APOBEC3 Proteins during HIV-1 Infection In the absence of HIV-1 Vif, APOBEC3 proteins interact with viral macromolecules at multiple stages of the HIV-1 life cycle to inhibit replicative processes resulting in nonproductive infection. In the target T cells, (1) a HIV-1Δvif virion with encapsidated A3G binds to coreceptors and receptors on the cell surface and traffics to the endosome. (2) The HIV-1 gp160 catalyzes the fusion of the viral and endosomal membranes to allow the release of its contents into the cytoplasm. (3) Reverse transcription (RT) occurs while the capsid is uncoating. Note: uncoating and reverse transcription are drawn as separate steps only for clarity in the figure. A3 oligomers bind the viral RNA and produce a roadblock to reverse transcriptase movement along the template, resulting in a reduction of viral DNA synthesis. The dissociation of A3 oligomers to monomers or dimers will rapidly bind to single-stranded DNA to inhibit HIV-1 replication through a deaminase-dependent mechanism. A3 proteins deaminate deoxycytidines to deoxyuridines in single-stranded DNA reverse transcription intermediates resulting in G → A hypermutation. In addition, A3 proteins are able to inhibit HIV-1 replication through deaminase-independent mechanisms by interacting with viral nucleocapsid (NCp7) to reduce tRNA priming and by directly interacting with HIV-1 reverse transcriptase resulting in reduced enzyme activity and processivity. Together these activities result in a decrease in the accumulation of HIV-1 reverse transcripts. (4) A3s are also able to interact with HIV-1 integrase to prevent proviral DNA formation and integration. In the producer cells following integration, the proviral genome is transcribed to produce progeny virion genomes, as well as templates for viral protein expression. (5) A3 proteins exist in the producer cell as active low-molecular mass (LMM) or inactive high-molecular mass (HMM) protein-RNA complexes. HIV-1 Vif recruits a E3-ubiquitin ligase complex, composed of EloB/C, cullin 5, core binding factor-β, and Rbx2, to ubiquitinate and target LMM A3 proteins for proteasomal degradation, thus preventing their encapsidation into nascent virions. (6) In the absence of Vif, LMM A3s interact with progeny virion genomes and Gag-associated nucleocapsid resulting in their encapsidation during viral assembly. Seven plus or minus four A3 molecules are incorporated per virion. Following (7) release and (8) maturation, the A3 carrying virion will go on to nonproductively infect a target cell. Structure 2014 22, 668-684DOI: (10.1016/j.str.2014.02.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 2 Structural Organization and Sequence Alignment of Human APOBEC3 Proteins (A) Domain organization of human A3 family members. Seven human A3 proteins (A3A, A3B, A3C, A3D, A3F, A3G, A3H) are encoded in a 150 kb region on chromosome 22. All A3 proteins contain at least one (A3A, A3C, and A3H) or two DNA cytosine deaminase catalytic domains (A3B, A3D, A3F, A3G). The three phylogenetic clusters of A3 Z domains, Z1, Z2, and Z3 are depicted as orange, blue and green, respectively. The Vif-binding A3 domains were indicated in the schematic. (B) Ribbon diagram of A3F C-terminal domain. The overall structural features of each DNA cytosine deaminase domains are very similar between human A3 family members. All A3 proteins adopt a common structural fold (α1-β1-β2-α2-β3-α3-β4-α4-β5-α5-α6). The crystal structure of the A3F-CTD (Protein Data Bank [PDB] codes: 4J4J and 4IOU) represents the Z2-type A3 domains, and it is catalytically active. The zinc ion at the catalytic site, shown in blue, has an important function during DNA cytosine deamination. The secondary structural elements are depicted as yellow for β sheet, magenta for α helices and light gray for random coil regions. (C) Multiple sequence alignment of A3 domains. Each domain of the double-domain A3 proteins (A3B, A3D, A3F, and A3G) is aligned separately and labeled as the N-terminal (NTD) or C-terminal (CTD) domain. Secondary structure prediction based on the Vif-binding A3F-CTD is depicted above the sequence alignment. Sequence numbering is based on A3A sequence shown at the top of the alignment. Similar residues are shown in red and outlined with white boxes. Residues that are strictly conserved in all A3 domains are highlighted with red boxes. The sequence alignment was performed by a Clustal W multiple sequence alignment (Sievers et al., 2011) and plotted using the program ESPript (Gouet et al., 1999). Structure 2014 22, 668-684DOI: (10.1016/j.str.2014.02.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 3 APOBEC3 HIV-1 Vif-Binding Sites (A) APOBEC3 proteins likely adopt three distinct types of Vif-binding sites: A3C/A3F-like, A3G-like, and A3H-like. Surface electrostatic potential and ribbon diagrams showing the three classes of A3 Vif-binding sites (yellow dashed lines) are shown. Negatively and positively charged regions are shown in red and blue, respectively. The A3C/A3F-like class is represented by A3C (PDB code: 3VOW), A3F-CTD (PDB codes: 4J4J and 4IOU) and A3D-CTD (homology model). The A3G-like and A3H-like classes are represented by homology models of the A3G-NTD and A3H, respectively. In A3C, A3F-CTD, and A3G-NTD, residues shown as red sticks in the ribbon diagrams represent experimentally determined Vif binding residues. Based on the A3F-CTD Vif-binding site, conserved residues are identified as red sticks in the A3D-CTD and A3H haplotype II homology models. Equivalent regions of the non-Vif binding A2 (PDB code: 2NYT) and A3A (PDB code: 2M65) show charge reversal and reduced hydrophobicity compared to the other A3 Vif-binding domains, thus potentially explaining its lack of Vif binding ability. (B) Schematic showing the conservation of critical residues involved in HIV-1 Vif binding for A3 proteins. The positions of secondary structural elements are shown in the figure. Experimentally determined Vif-binding residues are shown in A3C, A3F-CTD, and A3G-NTD, whereas in A3D-CTD and A3H only the equivalent residues that are conserved with A3F-CTD are shown (with the exception of E91 in A3H). Structure 2014 22, 668-684DOI: (10.1016/j.str.2014.02.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 4 HIV-1 Vif APOBEC3-Binding Sites (A) Structure of the HIV-1 Vif-CBF-β-EloB/C-CUL5 pentameric complex (PDB code: 4N9F). CBF-β, CUL5, and EloB/C are shown in a ribbon diagram, whereas HIV-1 Vif is depicted with its molecule surface. Defined HIV-1 Vif motifs previously shown to be necessary for A3G, A3F, or A3G/A3F binding are mapped onto Vif and colored in red, yellow and orange, respectively. The putative location of the missing A3G, C-terminal domain of CUL5, Rbx2, and E2 are shown as spheres. (B) Molecular surface of HIV-1 Vif illustrates two distinct binding surfaces for A3G and A3F. The A3F-binding surface is on the opposite side (180° away) from the A3G-binding interface. The presence of CBF-β on HIV-1 Vif partially restricts the A3F-binding site. The displacement of CBF-β may be necessary for the recruitment of A3F to the Vif-E3 ubiquitin ligase complex. The orientation of HIV-1 Vif in the left hand figure is in the same orientation as in (A). Structure 2014 22, 668-684DOI: (10.1016/j.str.2014.02.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 5 APOBEC3 DNA-Binding Sites Multiple models of single-stranded DNA binding have been observed in A3 proteins. NMR titration and X-ray crystal structures combined with mutagenic studies have revealed at least two models of DNA binding in the A3G-CTD. (A) A molecular surface of A3G-CTD (PDB codes: 3IR2, 2JYW, 3EIU, 2KBO) is presented with the directionality of the ssDNA of the two DNA binding models. A “kinked” model as proposed by Holden et al. (2008) is shown by a solid green line, whereas a “brim” or “straight” model, as proposed by Furukawa et al. (2009) and Chen et al. (2008) is displayed by a blue line. Key residues proposed by the brim or straight model are colored blue and white, respectively, while residues implicated in DNA binding by the kinked model are green. (B) In A3A (PDB code: 2M65), the ssDNA appears to bind along a “kinked” groove similar to the Holden et al. (2008) model in A3G-CTD. Experimentally identified residues that are important in A3 ssDNA binding are presented in Table 3. (C) In A3F-CTD (PDB code: 4J4J), ssDNA binds along a “straight” groove similar to the observed model by Furukawa et al. (2009) and Chen et al. (2008) in A3G-CTD. Structure 2014 22, 668-684DOI: (10.1016/j.str.2014.02.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 6 Deamination-Independent Interactions of APOBEC3G (A) Schematic representations of the HIV-1 Gag, RT, IN, and A3G proteins. Numbers above the schematics indicate the amino acid positions for each domain of the Gag, RT, IN, and A3G proteins. Regions in HIV-1 Gag, RT, and IN that interact with A3G are labeled in blue, orange, and green, respectively. The catalytic DNA cytosine deaminase domain of A3G is shown in red. The A3G residues responsible for dimer formation, DNA cytosine deamination, substrate specificity, and RNA, DNA, NC, RT, IN, Vif interactions are labeled on the A3G schematic. Abbreviations are as follows: Gag, group-specific antigen; RT, reverse transcriptase; IN, integrase; MA, matrix; CA, capsid; p2, spacer peptide 1; NC, nucleocapsid; p1, spacer peptide 2; p6, p6-Gag protein; NTD, N-terminal domain; CCD, core catalytic domain; CTD, C-terminal domain; A3G, APOBEC3G; CDD1, cytosine deaminase domain 1; CDD2, cytosine deaminase domain 2; Vif, viral infectivity factor. (B) Surface electrostatic potential and ribbon diagram of a homology model of the A3G-NTD. The A3G-NTD homology model was generated using the Phyre2 protein fold recognition server (Kelley and Sternberg, 2009). A negatively charged surface (showed in blue dash lines) was proposed to be the NCp7 binding site. The 128DPD130 motif involved in A3G Vif binding is highlighted with a yellow dashed line. A positively charged surface illustrates the footprint of the A3G-NTD residues (black dash lines) necessary for RNA binding and “head-to-head” dimer formation. (C) Molecular surface of the homology model of the A3G-NTD. HIV-1 RT and IN binding sites on A3G-NTD are colored as orange and green surfaces, respectively. Surfaces that overlap between the HIV-1 RT and IN binding sites are shown as a pink surface. Residues on the surface of A3G-NTD that may play a role in HIV-1 RT and IN interactions are shown as sticks colored according to the molecular surface. Interestingly, parts of the 128DPD130 Vif-binding residues and NCp7 binding site residues also converge on the overlap regions between the HIV-1 RT and IN binding sites (yellow dashed lines). Structure 2014 22, 668-684DOI: (10.1016/j.str.2014.02.011) Copyright © 2014 Elsevier Ltd Terms and Conditions