Abstract
The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.
Highlights
Human cells can express up to seven APOBEC3 (A3) proteins, which have the capacity to inhibit a broad number of endogenous and exogenous retroelements including viruses such as HIV-1 [1,2,3,4,5]
These A3A-double-stranded DNA (dsDNA) complexes were termed non-specific, because prior studies demonstrated that A3A only deaminates single-stranded DNA (ssDNA) cytosines [19] and the yield of these complexes was lower (,20%)
The hybrid DNA approach developed in our previous publications allow us directly observe the binding of A3A protein with ssDNA, and with dsDNA
Summary
Human cells can express up to seven APOBEC3 (A3) proteins, which have the capacity to inhibit a broad number of endogenous and exogenous retroelements including viruses such as HIV-1 [1,2,3,4,5]. A3A is not a strong inhibitor of Vif-deficient HIV-1 in T cell lines [18]. It may have an antiviral role in myeloid lineage cell types because it is strongly induced by type 1 interferons and other innate immune agonists [2,8,10], and it can trigger the clearance of naked double-stranded DNA (dsDNA) by a deamination and uracil excision-dependent mechanism [10]. Further consistent with a role in innate immunity, A3A has been shown to suppress the replication of LINE-1 [9], AAV-2 [6], and HTLV-1 [3,8,9] in model systems
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