A DNA Sequence Recognition Loop on APOBEC3A Controls Substrate Specificity

<div><p>APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active <i>E. coli</i>-produced A3A. The enzyme binds to single-stranded DNA with a K_d of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.</p></div

The preferred consensus deamination target site of A3A is TC (A/G).

<p>A) rA3A was incubated with ssDNA substrates containing four different target site sequences. B) rA3A was incubated with ssDNA substrates containing different target site sequences.</p

Type-I and type-II IFN induce A3A in monocytes, MDM and MDDC.

<p>Human monocytes, MDM, MDDC and activated CD4+ T cells were treated with IFN-β or IFN-γ. After 24 h the cells were lysed and protein levels determined by immunoblot using a rabbit antiserum specific for A3A. Similar results were obtained with cells purified from the blood of three independent donors.</p

Residues in the putative DNA binding cleft determine binding to ssDNA.

<p>A) The A3A model predicts four conserved amino acids that could play a role in DNA binding. Each was mutated to alanine. B) The mutated A3A proteins were expressed in transfected 293T cells, immunoprecipitated from cell lysates and tested for deaminase activity against a deoxyoligonucleotide DNA containing a T<u>C</u>G target site. C) <i>E. coli</i> expressed mutated A3A proteins in the background of the E72A catalytic site mutation were purified and similar amounts of each were analyzed for ssDNA binding in the gel retardation assay. D) A fluorescein tagged oligonucleotide was incubated with increasing amounts rA3A from (C). The change in the extrinsic fluorescence at each protein concentration was used to fit a one-site saturation ligand-binding curve. The calculated K_d for each curve is displayed on a table together with the standard error. ND indicates that a K_d could not be determined.</p

A3A deamination is nonprocessive.

<p>A) An oligonucleotide containing a fluorescein tag flanked by consensus deamination sites was incubated with rA3A. B) The oligonucleotide was incubated with rA3A for 2, 5, 10 and 20 minutes, and single or double deamination at the 5′- and 3′-target sites was determined. C) The processivity factor was calculated for each time point. D) An oligonucleotide containing a series of target sites was incubated with rA3A and deamination states were identified by DNA sequencing. E) The frequency of deaminations at sequential target sites were quantified. Eight of the sequenced oligonuleotides contained no mutations and are indicated as having 0 sequential deamination sites.</p

A3A homodimerizes <i>in vitro</i> and <i>in vivo</i>.

<p>A) <i>top.</i> rA3A was run on a size exclusion column. <i>bottom.</i> A3A content was tested for each fraction by immunoblot using the anti-A3A antibody. B) The overall protein content of each fraction was determined by Bradford assay, and catalytic activity was determined by <i>in vitro</i> deaminase assay with a biotinylated TCA oligo using an equal volume of each fraction. C) 293T cells were transfected with expression vectors for HA, FLAG-tagged A3A or both. The cells were lysed, and the HA-tagged A3A was immunoprecipitated. The immunoprecipitates were then analyzed on an immunoblot.</p

rA3A produced in <i>E. coli</i> binds and deaminates ssDNA.

<p>A) rA3A was visualized by Coomassie staining B) The extent of deamination was determined for rA3A incubated with ssDNA for 2, 5, 10, 20, 40, or 60 minutes. C) DNA binding was determined by measuring the change in the intrinsic fluorescence of rA3A following incubation with an increasing amount of ssDNA.</p

The target site preference of A3A is influenced by RL1.

<p>A) A model of A3A on the A3G crystal structure shows loops RL1 and RL2 (in red) flanking the proposed DNA binding groove and shows the inserted the WG amino acids (in orange) close to the conserved catalytic E72 (in green). Mammalian expression vectors for A3A in which RL1 or RL2 of A3G was swapped into A3A (RL1 and RL2) and a mutated A3A deleted for the WG were generated. B) The mutated proteins were expressed in transfected 293T cells, immunoprecipitated from cell lysates and then tested for deaminase activity against an oligonucleotide containing a T<u>C</u>G target sequence. C) The deamination activity of the mutated proteins was expressed as the ratio of the intensity of the deaminated product to the sum of the intensities of the unmodified and deaminated species. D) The target site specificity of the immunoprecipitated proteins was determined by incubating with oligonucleotides containing an N<u>C</u>N target site. The relative percentage deamination was determined as the percent deamination of the NCN oligonucleotide normalized to the T<u>C</u>A oligonucleotide. E) The target site specificity was determined as the ratio of deaminase activity determined for the N<u>C</u>s oligonucleotides to the total deamination activity.</p

Patient-derived monoclonal antibody neutralizes SARS-CoV-2 Omicron variants and confers full protection in monkeys.

The SARS-CoV-2 Omicron variant has very high levels of transmission, is resistant to neutralization by authorized therapeutic human monoclonal antibodies (mAb) and is less sensitive to vaccine-mediated immunity. To provide additional therapies against Omicron, we isolated a mAb named P2G3 from a previously infected vaccinated donor and showed that it has picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other variants tested. We solved the structure of P2G3 Fab in complex with the Omicron spike using cryo-electron microscopy at 3.04 Å resolution to identify the P2G3 epitope as a Class 3 mAb that is different from mAb-binding spike epitopes reported previously. Using a SARS-CoV-2 Omicron monkey challenge model, we show that P2G3 alone, or in combination with P5C3 (a broadly active Class 1 mAb previously identified), confers complete prophylactic or therapeutic protection. Although we could select for SARS-CoV-2 mutants escaping neutralization by P2G3 or by P5C3 in vitro, they had low infectivity and 'escape' mutations are extremely rare in public sequence databases. We conclude that this combination of mAbs has potential as an anti-Omicron drug