APOBEC3H Antiviral Mechanisms Driven by Protein-Nucleic Acid Interactions: Structure and Function

Abstract

Human Immunodeficiency Virus (HIV) is a retrovirus that stores its genetic information as ribonucleic acid (RNA). Reverse transcription converts the genome to a stable double-stranded deoxyribonucleic acid (DNA) intermediate which the virus integrates into the host genome, a feature of HIV that makes it difficult to treat. Interferon response pathways trigger the expression of host proteins known as restriction factors, which hinder HIV replication. The APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3) family is composed of seven (A3A-A3H) conserved zinc-dependent cytidine deaminases, three of which (APOBEC3F, APOBEC3G, and APOBEC3H) exhibit restriction activity against HIV. A3F and A3G consist of two zinc-binding domains, both of which are required for restriction activity, while A3H uses just one domain. Through a broad set of cellular, biochemical, and structural approaches, this thesis work presents a full characterization of a model restriction factor, APOBEC3H. Antiviral APOBEC3 proteins function through a series of interactions with a diverse set of viral nucleic acid species. In an infected cell, APOBEC3 proteins bind to viral RNA to become encapsidated into budding virions, however the mechanism by which APOBEC3 proteins select viral RNA over cellular RNA was largely unknown. While previous reports suggested that A3 proteins nonspecifically bind to single-stranded regions of RNA, here, the structural characterization of an A3H-RNA complex revealed that A3H surprisingly recognizes and binds to segments of A-form duplex RNA in vitro and in virions. This important finding explains the selective binding of A3H to the viral RNA as the HIV genome is comprised of many structured elements and is compacted during virus assembly, thus binding to RNA duplexes allows A3H to recognize these structures to infiltrate the virus for downstream restriction activities. Detailed dissection of the A3H-RNA interface highlights the importance of A-form duplex binding in the remainder of A3H antiviral activities, which include inhibition of reverse transcription and cytidine deamination of the single-stranded viral DNA product of reverse transcription. How does the single domain of A3H sense both RNA and DNA when A3F and A3G appear to use separate domains for RNA and DNA recognition? In this work, comprehensive mechanistic characterization of A3H substrate selection revealed that A3H not only deaminates ssDNA substrates but can bind RNA/DNA heteroduplexes and deaminate associated ssDNA overhangs, suggesting that the RNA binding mode of A3H also applies to catalysis. This analysis broadens the substrate scope of A3H given that RNA/DNA hybrids with ssDNA overhangs exist during reverse transcription. Restriction of HIV by APOBEC3 proteins is highly successful, but only in the absence of the viral accessory protein known as Vif, which targets A3 proteins for proteasomal degradation by hijacking the host ubiquitin ligase machinery. The molecular basis for the species-specific targeting of A3 proteins by Vif may be driven through electrostatics, but the details of these interactions remain mysterious. Biochemical tools were developed for expression and purification of host-virus protein complexes, such as A3-Vif. These materials are critical for future structural investigations to understand the interactions that allow the virus to evade host defenses. This thesis work paints a detailed portrait of the mechanistic underpinnings of viral restriction for APOBEC3H and lays the groundwork for future structural characterization of A3-Vif interactions, which are critical for viral pathogenesis.