Apobec3G Transitions from a Mobile to Static Binding State through Dimerization Mediated by the N-Terminal Cytidine Deaminase Domain

Abstract

Listen

Translate article

APOBEC3G (A3G) is a human enzyme with anti-viral properties and is a particularly potent inhibitor of HIV-1 infectivity in the absence of viral infectivity factor. A3G is a deoxycytidine deaminase which creates C to U base mutations on HIV minus strand DNA during replication, reducing viral viability. However, A3G can still inhibit HIV infectivity even in the absence of deaminase activity. It has been proposed that A3G bound to minus stand viral DNA physically blocks reverse transcription of the complementary plus strand and that A3G's ability to form this roadblock depends on its ability to form oligomers while bound to DNA. Using single molecule methods, we measure the kinetics of A3G binding to single-stranded DNA and the subsequent formation of bound A3G oligomers. We repeat these experiments using A3G variants with mutated cytidine deaminase domains in order to determine the role of each of the two domains in oligomer formation. We determine that A3G first transiently binds DNA as a monomer then forms dimers through interactions between the N-terminal domains. While monomers freely slide along DNA and dissociates entirely on the timescale of a minute, dimerization prevents sliding and increases binding time by more than an order of magnitude. Our results suggests viral DNA deamination is performed by monomeric A3G while reverse transcription is inhibited by the formation of A3G dimers. This new model helps explain how a small number of A3G subunits packaged in the virion can both rapidly deaminate viral DNA and disrupt reverse transcription on two greatly different timescales.