Oligomerization of HIV-1 Restriction Factor APOBEC3G Transforms it from a Fast Enzyme to a Slow Nucleic Acid Binding Protein

Abstract

Human APOBEC3G (A3G) is a cellular protein that inhibits reverse transcription and replication of human immunodeficiency virus type-1 (HIV-1) in the absence of the viral protein Vif. A3G impairs viral replication by two different mechanisms, which both rely on its ability to bind single-stranded nucleic acids. First, A3G deaminates cytidine bases of viral single-stranded DNA (ssDNA). Secondly, A3G blocks DNA synthesis by reverse transcriptase (RT), the viral DNA polymerase, by a mechanism independent of catalytic activity. Seven A3G proteins are packaged per HIV-1 virion, requiring that each molecule rapidly locate deamination sites on viral ssDNA, which is a transient intermediate during reverse transcription. In contrast, the roadblock mechanism, a model in which A3G oligomerizes on the viral template strand and blocks RT-catalyzed DNA elongation, requires an extremely slow off-rate from single-stranded nucleic acids. We hypothesize that A3G exhibits fast binding kinetics as a dimer, enabling rapid deamination activity, and slow kinetics as an oligomer, preventing RT from elongating viral DNA. We use optical tweezers, in combination with fluorescence anisotropy and surface plasmon resonance, to quantify both types of binding kinetics. DNA stretching experiments reveal that the time constant for oligomerization, ranging from 200 to 1000 s, is inversely dependent on protein concentration. The apparent dissociation constant of A3G oligomerization decreases exponentially with ssDNA incubation time, dropping by an order of magnitude in 1000 s, which suggests that fast binding of catalytically active dimers converts to oligomerization on this timescale. This slow association and dissociation of A3G oligomers, which is consistent with ensemble methods, supports the roadblock hypothesis. Collectively, our measurements quantitatively characterize the complex, highly unusual nucleic acid binding kinetics of A3G responsible for its dual mechanism for inhibiting viral replication.