Mapping the DNA and RNA Binding Domains of the HIV Restriction Factor APOBEC3G

Abstract

APOBEC3G (A3G) is an ssDNA binding protein and HIV restriction factor. A3G mutates the viral genome during reverse transcription through deamination of dC to dU. The C‐terminal catalytic domain only supported ssDNA binding and deaminase activity within the full length protein. Cellular RNAs inhibit A3G binding and deamination of ssDNA, and form ribonucleoprotein complexes with A3G. Point mutations mapped RNA binding to the N‐terminus but again only full‐length A3G bound RNA. We evaluated quaternary complexes of A3G bound to ssDNA and RNA using near equilibrium binding, gel shift analyses and fluorescence anisotropy. The domains of A3G in contact with ssDNA or RNA were found by UV crosslinking and mass spectral sequencing. The data showed a lax ssDNA and RNA sequence requirement for A3G binding, but oligonucleotides below 15 nt showed impaired binding. A3G bound ssDNA as a homodimer that became catalytically active upon the formation of A3G homotetramers as a highly ordered process. RNA formed differently sized complexes with A3G. RNA dissociated ssDNA from A3G and simultaneously disrupted dimers and tetramers of A3G. Mass spectrometry demonstrated that ssDNA and RNA bound primarily unique A3G peptides dispersed along the entire protein. The data suggested a mechanism for the assembly of A3G catalytic complexes that explains the processivity of mutagenesis through dimer and tetramer formation. We predict the stochastic dissociation of A3G:ssDNA complexes by RNA is due to both conformational changes from RNA binding to allosteric sites not occupied with ssDNA as well as competitive binding. The work was supported by grants awarded to HCS from NIAID 095007 and NIGM 110568.