The Binding Interface between Human APOBEC3F and HIV-1 Vif Elucidated by Genetic and Computational Approaches

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV-1 Vif protein, which subverts cellular CBF-β and a polyubiquitin ligase complex to neutralize these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host-pathogen conflict has been elusive. We combine virus adaptation and computational studies to interrogate the APOBEC3F-Vif interface and build a robust structural model. A recurring compensatory amino acid substitution from adaptation experiments provided an initial docking constraint, and microsecond molecular dynamic simulations optimized interface contacts. Virus infectivity experiments validated a long-lasting electrostatic interaction between APOBEC3F E289 and HIV-1 Vif R15. Taken together with mutagenesis results, we propose a wobble model to explain how HIV-1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, how pathogens may evolve to escape innate host defenses

Natural Polymorphisms in Human APOBEC3H and HIV-1 Vif Combine in Primary T Lymphocytes to Affect Viral G-to-A Mutation Levels and Infectivity

<div><p>The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host <i>APOBEC3</i> genes and the viral <i>vif</i> gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human <i>APOBEC3</i> genes is <i>APOBEC3H</i> with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as <i>in vivo</i> barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.</p></div

Stable APOBEC3H inhibits HIV-1 replication in primary T lymphocytes and inflicts GA-to-AA hypermutations.

<p>A) HIV-1 replication kinetics of the hyper-, lab-, and hypo-Vif variants in CD4+ T lymphocytes from a representative healthy donor encoding unstable A3H haplotype I/I (donor 2). Data from additional experiments using independent donors are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004761#pgen.1004761.s003" target="_blank">Figure S3</a>. Y-axis values represent p24 levels measured by ELISA. B) HIV-1 replication kinetics of the hyper-, lab-, and hypo-Vif variants in CD4+ T lymphocytes from a representative healthy donor encoding one allele of stable A3H haplotype II (and one allele of unstable A3H haplotype I; donor 4). Data from additional experiments using independent donors are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004761#pgen.1004761.s003" target="_blank">Figure S3</a>. Y-axis values represent p24 levels measured by ELISA. C) Immunoblots of the indicated proteins in non-normalized virus-like particle (VLP) supernatants and in cells from days 3, 5, 7, and 9 of the spreading experiments shown in panels A & B. A quantification of the ratio of A3H to CA is shown below each VLP blot set. D) 3D-PCR amplicons generated from proviral DNA of the indicated viruses isolated on day 15 of a spreading infection of unstable A3H (haplotypes I/I and III/IV) or stable A3H (haplotype I/II) donor cells (donors 2, 4, 12). This experiment is representative and performed independently of those shown in panels A-C. E) Histograms depicting the frequencies of GA-to-AA and GG-to-AG mutations under the indicated spreading infection conditions (complementary to the experiment shown in panel D with all sequences derived from independent 98°C high-fidelity PCR amplifications). A minimum of 10 clones were sequenced for each condition (≥5 kb).</p

Generation and validation of HIV-1 Vif separation-of-function molecular/viral probes.

<p>A) A schematic of the Vif protein encoded by each HIV-1 molecular clone showing amino acid differences responsible for the hyper- and hypo-Vif functionality relative to lab-Vif (HIV-1 IIIB/NL4-3) against stable A3H haplotype II. B) Immunoblots showing the expression levels of the indicated A3 proteins stably expressed in SupT11 cells. In this experiment untagged A3H is detected with the mouse monoclonal antibody P3A3-A10. C) HIV-1 spreading infection kinetics for the indicated viruses on A3-expressing SupT11 cells lines described in panel B. The hyper-, lab-, and hypo-Vif isolates spread with similar kinetics on cells expressing a control vector, A3D, A3F, or A3G, but showed clear phenotypic differences on cells expressing low, intermediate (int), and high levels of stable A3H haplotype II. Delta-Vif virus replication was evident in control vector expressing SupT11 cells, delayed in A3D expressing cells, and suppressed under all other conditions (some symbols eclipsed).</p

Endogenous APOBEC3H stability/instability occurs at the protein level.

<p>A) A schematic of the 7 gene <i>A3</i> locus and the 5 polymorphisms in <i>A3H</i> exons 2, 3, and 4 that combine to produce 7 different haplotypes. On the right, a summary of the 7 different A3H haplotypes based on observed protein stability or instability in overexpression studies <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004761#pgen.1004761-OhAinle1" target="_blank">[35]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004761#pgen.1004761-Wang1" target="_blank">[36]</a> and as defined here, in primary CD4+ T lymphocytes. The two residues that underlie the stable/unstable phenotypes are highlighted in red (N15/Δ15 and R105/G105). B) Immunoblots showing endogenous A3H, A3G, and HSP90 protein levels in stimulated primary T lymphocytes from 4 donors with the indicated <i>A3H</i> haplotypes (donors 10, 11, 12, and 18). In this experiment endogenous A3H is detected with a polyclonal rabbit antibody. C) <i>A3</i> mRNA levels in primary T lymphocytes from 4 donors with the indicated <i>A3H</i> haplotypes (donors 4, 10, 12, and 18). Expression levels are shown relative to the housekeeping gene <i>TBP</i>.</p

Correlations between the global distributions of HIV-1 hyper-Vif alleles and human A3H haplotypes.

<p>A) The left histogram depicts the frequency of HIV-1 isolates encoding a phenylalanine or valine at Vif residue 39 from the indicated geographic regions (n = 9713; <a href="http://www.hiv.lanl.gov" target="_blank">www.hiv.lanl.gov</a>). The right histogram shows the frequency of stable versus unstable <i>A3H</i> alleles from the same geographic regions (n = 1092; <a href="http://www.1000Genomes.org" target="_blank">www.1000Genomes.org</a>). B) A model depicting the anticipated relative transmission efficiencies between infected patients and uninfected individuals with equivalent or different <i>A3H</i> haplotypes.</p

Vif separation-of-function substitutions define the likely APOBEC3H interaction surface.

<p>A) A surface representation of Vif and CBFβ (pdb 4N9F). The side-chains of the amino acid residues conferring hypo-Vif (residue 39) and hyper-Vif phenotypes (residues 48 and 60–63) are shaded green and located on a common solvent-exposed surface. A 90° rotation reveals distinct Vif separation-of-function residues implicated in the interactions with A3F (yellow) and A3G (orange). See main text for details. B) Spreading infection kinetics of the indicated HIV-1 Vif variants on SupT11 cells stably expressing a vector control (left) or high levels of A3H haplotype II (right). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004761#pgen-1004761-g004" target="_blank">Figure 4C</a> data for hyper-, lab-, and hypo-Vif are shown again here to facilitate comparisons.</p

APOBEC3H adaptation studies.

<p>A) Immunoblots of A3H and tubulin in SupT11 cell lines stably expressing low, intermediate (int), and high levels of stable haplotype II protein. In this experiment A3H is detected with the mouse monoclonal antibody P3A3-A10. B) Schematic of the stepwise A3H adaptation procedure (see text and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004761#s4" target="_blank"><b>Materials and Methods</b></a> for details). C) Spreading infection kinetics of HIV-1 molecular clones encoding the indicated Vif proteins on the A3H-expressing SupT11 lines shown in A.</p

Structure of the Vif-binding domain of the antiviral enzyme APOBEC3G

The human APOBEC3G (A3G) DNA cytosine deaminase restricts and hypermutates DNA-based parasites including HIV-1. The viral infectivity factor (Vif) prevents restriction by triggering A3G degradation. Although the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, we used evolution- and structure-guided mutagenesis to solubilize the Vif-binding domain of A3G, thus permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish the structure from previous catalytic-domain structures and help to explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by Vif; this enabled mutagenesis and biochemical experiments, which identified a unique Vif-interacting surface formed by the alpha1-beta1, beta2-alpha2 and beta4-alpha4 loops. This structure sheds new light on the Vif-A3G interaction and provides critical information for future drug development