APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4°C did not have significant deaminase activity, but their enzymatic function could be restored by subsequent incubation at 37°C. This approach enabled complexes of a defined size range to be isolated and subsequently evaluated for their contribution to enzymatic activity. The composition of A3G bound to ssDNA was determined by protein-protein chemical cross-linking. A3G-ssDNA complexes of 16 S were necessary for deaminase activity and consisted of cross-linked A3G homotetramers and homodimers. At lower concentrations, A3G only formed 5.8 S homodimers on ssDNA with low deaminase activity. Monomeric A3G was not identified in 5.8 S or 16 S complexes. We propose that deaminase-dependent antiviral activity of A3G in vivo may require a critical concentration of A3G in viral particles that will promote oligomerization on ssDNA during reverse transcription.APOBEC3G is a cytidine deaminase whose expression has been implicated in host defense and conditionally in viral resistance (1). The APOBEC 4 protein family includes APOBEC1 (after which the family is named), activation-induced deaminase, APOBEC2, APOBEC3A-H, and APOBEC4 (2-5). The catalytic activity of these enzymes involves hydrolytic deamination of cytidine to form uridine or deoxycytidine to form deoxyuridine in single-stranded RNA or single-stranded DNA. There is considerable interest in this class of proteins because they are capable of mutagenic activity and epigenetic regulation of protein variant expression that can influence tissue functions and disease progression (4, 6).APOBEC proteins are characterized by a helix-strand-helix supersecondary structure containing conserved residues (His/ Cys)-Xaa-Glu-Xaa 25-30 Pro-Cys-Xaa-Xaa-Cys that are integral to the function of the zinc-dependent deaminase domain or ZDD (5). Several of the APOBEC proteins, such as APOBEC1 and activation-induced deaminase, contain a single ZDD that was responsible for catalytic activity. Other family members, such as APOBEC3B and APOBEC3G, contain multiple ZDDs within a single molecule where both domains or a single domain were catalytically active (2, 3).The N-terminal and C-terminal ZDD in A3G were required for nucleic acid-dependent higher-order oligomerization as well as many other interactions necessary for antiviral activity and wild type levels of deaminase activity (7-11). In isolation, the N-terminal ZDD could not support RNA or ssDNA binding (12), but the C-terminal half of A3G supported a modest level of deaminase activity (12-16).The crystal structure of full-length APOBEC2 and lower resolution small angle x-ray scattering structure of native, fulllength A3G revealed ...