Structural and Mechanistic Insight into APOBEC3G DNA Binding and Deamination

Abstract

Humans express the APOBEC3 family of proteins to defend against endogenous and exogenous DNA pathogens. APOBEC3 proteins display significant activity towards HIV-1 through incorporation into budding viral particle and interacting with HIV's RNA genome. In order to stably integrate into the human genome, HIV reverse transcribes the single stranded RNA genome into a double stranded DNA genome through a single stranded DNA intermediate. Once bound to the transient single stranded viral DNA intermediate, APOBEC3 proteins deaminate cytidines to uridines. Transcription over the resulting mutations results in G to A transversions in the coding sequence of the virus and non-functional gene products. APOBEC3 proteins are highly active on the single stranded DNA intermediate but lack catalytic activity on cytidines present in the viral RNA. APOBEC3G is the most potent of these innate viral mutagens and selectively targets tri-cytidine hotspot motifs in viral genome intermediates. When I began my thesis research, the effects of A3G mutagenesis had been extensively studied but the structural interactions with single stranded DNA and the mechanism of RNA exclusion were unknown. My thesis research helped identify the amino acid responsible for pH dependent effects on substrate binding. The identification of this residue allowed for the development of a pH insensitive variant of A3G that provided indirect evidence for a reduced pH in the viral capsid during the initiation of reverse transcription. These results, along with the kinetic characterization of A3Gctd and determination of the substrate factors crucial for deamination, are described in Chapter 2. In chapter 3, I continued to explore the effects of this pH dependent increase in binding affinity. I utilized NMR spectroscopy to identify the structural interactions of catalytic substrate binding. By generating a structurally stable but catalytically inactive mutant, I was able to identify differences between substrate interactions. This mutant also allowed me to explore structural interactions involved in substrate recognition and RNA exclusion. This lead to the identification of a novel substrate and a ribose sugar pucker dependent mechanism for target discrimination. The role of APOBEC3 proteins in retroviral restriction as well as their connection to several types of cancer makes them a prime target for therapeutic interventions. My thesis research in trying to understand the structural and mechanistic components of the APOBEC3/DNA interaction provides information that may be useful in the development of treatments targeting this family of proteins.