Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

Abstract

APOBEC3G (A3G) is a potent antiretroviral deoxycytidine deaminase that, when incorporated into HIV virions, hypermutates nascent viral DNA formed during reverse transcription. HIV Vif counters the effect of A3G by depleting intracellular stores of the enzyme, thereby blocking its virion incorporation. Through pulse-chase analyses, we demonstrate that virion A3G is mainly recruited from the cellular pool of newly synthesized enzyme compared to older “mature” A3G already residing in high-molecular-mass RNA–protein complexes. Virion-incorporated A3G forms a large complex with viral genomic RNA that is clearly distinct from cellular HMM A3G complexes, as revealed by both gel filtration and biochemical fractionation. Unexpectedly, the enzymatic activity of virion-incorporated A3G is lost upon its stable association with HIV RNA. The activity of the latent A3G enzyme is ultimately restored during reverse transcription by the action of HIV RNase H. Degradation of the viral genomic RNA by RNase H not only generates the minus-strand DNA substrate targeted by A3G for hypermutation but also removes the inhibitory RNA bound to A3G, thereby enabling its function as a deoxycytidine deaminase. These findings highlight an unexpected interplay between host and virus where initiation of antiviral enzymatic activity is dependent on the action of an essential viral enzyme. APOBEC3G (A3G) is a cellular enzyme that promotes DNA mutagenesis and can restrict infection by HIV-1. However, HIV counters the antiviral effects of A3G through the action of its Vif protein. In the absence of Vif, A3G is effectively incorporated into virions, where it mutagenizes the first DNA copy (cDNA) generated during reverse transcription of the viral RNA genome. A3G also appears to be able to inhibit HIV via nonenzymatic mechanisms. A3G and related deoxycytidine deaminases can also inhibit the growth of retroviruses other than HIV and protect the cellular genome from endogenous mobile retroelements. In this study, we analyzed the recruitment and enzymatic activity of A3G incorporated into HIVΔVif virions. Unexpectedly, we found that the binding of A3G to viral genomic RNA led to inactivation of the enzyme. However, latent A3G was ultimately activated through the action of HIV RNase H, which degrades the RNA genome during reverse transcription. These findings highlight an unexpected interplay between a host enzyme and HIV, where the antiviral enzymatic activity of the host factor (A3G) is dependent on the action of an essential HIV enzyme (RNase H). The strong interaction with viral RNA also suggests a potential mechanism by which A3G could exert antiviral activity in the absence of enzymatic activity, by physically impeding reverse transcription.