Local sequence targeting in the AID/APOBEC family differentially impacts retroviral restriction and antibody diversification

Abstract

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T, R = A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

FIGURE 1.

Levels of targeting by AID/APOBEC enzymes. A, global and local targeting. APOBEC3 enzymes, used by the innate immune system for retroviral restriction, are globally targeted to the HIV virion and show a local preference for deamination of cytidine residues following a pyrimidine. The viral accessory protein Vif prevents global targeting by promoting the degradation of the host APOBEC defense factor. AID, the master catalyst governing somatic hypermutation and class switch recombination, is globally targeted to the immunoglobulin locus and locally targeted to WRC sequences within the variable locus and switch regions. B, loop grafting. An alignment of AID, A3G, and A3F demonstrates a region of sequence divergence, the hotspot recognition loop (*), flanked by highly conserved sequences. Grafting of the loop between AID/APOBEC3 family members offers a means to probe the importance of local sequence targeting upon retroviral restriction, somatic hypermutation, and class switch recombination.

FIGURE 2.

Loop graft variants of A3G effectively restrict HIV despite altered local sequence targeting. A, global targeting is preserved with loop graft variants. After SDS-PAGE on purified pseudoviruses, blots were probed with monoclonal anti-His and anti-p24 antibodies demonstrating that A3G-WT, A3G-3FL, A3G-AL, and A3G-E259A are all packaged similarly into virus particles. B, viral cDNA production is modestly impacted by A3G incorporation. Real time PCR performed with primers specific for late cDNA transcripts was used to quantify the total viral cDNA after infection of Jurkat cells. Measurements from 5–7 replicate infections were combined to obtain mean copies and S.E. C, loop graft variants are effective in retroviral restriction. Increasing amounts of virus were used to infect Jurkat cells, which were assessed for GFP expression. The fraction of GFP-positive cells and S.E. from infections with three independent virus preparations is shown. D, grafting a foreign loop alters local sequence targeting. Local sequences in the region from −4 to +4 relative to the target mutation were catalogued. The probability logos in the (−)-strand cDNA target are shown (C → U transition at position 0). The most probable residue at each position is indicated along the top. The height of each residue corresponds to the probability of its occurrence at a given position, with the exception of position 0.

FIGURE 3.

Targeted hypermutation at CDRs is perturbed by loop graft mutants. A, targeting of CDRs is influenced by loop grafting. From transfected AID^−/− UNG^−/− DT40 B-cells propagated under selection and sorted for loss of IgM expression, the V_λregion was amplified and sequenced. The frequency of mutations found at each position in V_λ is shown, with the CDRs highlighted. B, AID variants alter the local sequence targeting of hypermutation. The local DNA sequence in the region from −4 to +4 relative to the target mutations were catalogued. The sequences were read relative to the sense strand for C → T mutations and in the antisense strand for G → A mutations. The probability logo is shown (target C → U at position 0). The most probable residue at each position is indicated along the top. The height of each residue corresponds to the probability of its occurrence at a given position, with the exception of position 0.

FIGURE 4.

Frequency of class switch recombination is proportional to AID hotspot targeting. A, expression of AID variants by Western blot. After SDS-PAGE on protein extracts from AID^−/− splenic B-cells infected with retroviruses expressing AID variants, the blots were probed with polyclonal anti-AID and anti-β-actin antibodies to demonstrate that AID-WT, AID-3FL, AID-3GL, and AID-E58A are expressed similarly. B, class switching is influenced by loop grafting. After infection, B-cells were stimulated to promote CSR from IgM to IgG1. Shown is a representative dataset gating on GFP expression as a marker of infection and IgG1 expression as a measure of class switching from IgM. C, AID variants that less efficiently target WRC sequences compromise CSR. Switching was calculated as the IgG1⁺ GFP⁺ population compared with the total GFP⁺ population with S.E. from three replicates shown.