Understanding Nucleic Acid Interactions of the Anti-HIV Protein APOBEC3G
Lauren Holden, University of Southern California, Los Angeles
Advisor: Xiaojiang Chen
Training in Basic Biomedical Sciences
Dissertation Award
2009
The Apolipoprotein B editing enzyme catalytic polypeptide-like (APOBEC) family of proteins deaminate cytidines on either single-stranded DNA (ssDNA) or RNA substrates, inducing C to U mutations that are exploited for a variety of biological purposes. This activity is controlled by a signature domain motif His-Xaa-Glu-Xaa23-28-Pro-Cys-Xaa2-4-Cys, where the histidine and two cysteine residues coordinate the Zn required for catalysis. A duplication event occurred during evolution that produced four APOBECs with two of these domains. One such member is APOBEC3G (A3G). In A3G, it has been shown that only the second domains (CD2) is catalytically active, while the other is responsible for RNA binding and various protein interactions.
A3G has been shown to potently restrict HIV replication. A3G introduces multiple dC to dU deaminations in the viral cDNA during reverse transcription, triggering degradation of the viral cDNA or resulting in defective proviruses. A3G also appears to interfere with successful infection by HIV in a non-deamination dependent manner. To counteract this antiviral activity of A3G, HIV-1 encodes the Vif protein that binds and targets A3G for proteosome-mediated degradation. However, in the absence of Vif, A3G can effectively restrict HIV-1 replication. Over the past several years, there has been great progress towards a biochemical understanding of these processes, including our structure of CD2. However several questions still remain: What structural features are governing these protein-nucleic acid and protein-protein interactions? Why is there a division of labor between homologous domains within a single protein? How is that segregation established? How can this be used to design effective drug targets?
In this study I propose two simple approaches to answering these questions: (1) co-crystallization of CD2 with ssDNA substrates and (2) crystallization of full length A3G. Determination of the molecular interactions between A3G and nucleic acid substrates will facilitate our understanding of the deamination dependent anti-viral action of A3G, while the atomic structure of full-length A3G will provide a context from which better insights into the interactions with Vif can be drawn. These insights will, in turn, allow us to develop new strategies to protect A3G from Vif targeted degradation, while simultaneously preserving the anti-viral action of A3G.
Altogether, this study will provide invaluable information for the fight against HIV by laying a foundation for novel drug discovery treatments.
