Single-Stranded DNA Binding and Crosslinking Activities of the Viral Restriction Factor Apobec3G Characterized by Force Spectroscopy

Abstract

APOBEC3G (A3G) is a human deaminase able to inhibit human immunodeficiency virus type 1 (HIV-1). A3G reduces HIV-1 infectivity via two independent mechanisms, including enzymatic hyper-mutation of the viral genome, and non-enzymatic stalling of the reverse transcription. However, the physical mechanism of these A3G activities remain unclear. Here we use force spectroscopy to show that the ssDNA-bound A3G exists in the rapid (1-10 s) conformational equilibrium between its “globular” state with its NTD and CTD bound to each other, with free energy ∼2.4 kBT, and its extended “dumbbell” state with its domains unbound, but connected by ∼0.6 nm flexible 7 aa linker. Low free energy and fast kinetics of this conformational transition in ssDNA-bound A3G suggest that both of these states are functionally important. We also measure much slower rates of monomeric A3G binding to and unbinding from ssDNA, which are of 107 M−1.s−1 and 0.014 s−1, respectively. However, while all of dimerization deficient FW A3G mutant dissociates with that rate, the a majority of the WT A3G oligomerizes upon binding within 10 s, and does not dissociate for over 10 min. We show that the A3G monomers can crosslink two DNA strands, leading to major construct shortening. For the WT A3G oligomerized on ssDNA the strand crosslinking process takes ∼300 s, while it only takes ∼10 s for the FW A3G mutant. In both cases crosslinking is rate-limited by one of the A3G domain unbinding one ssDNA strand and re-binding another strand. Taken together with other published data, our results suggest that the ssDNA-crosslinking A3G monomers are calalyticalycatalytically active, while the oligomerized A3Gs are catalytically inactive, and present a reverse transcription road block.