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Studying the Accessory Proteins and Functions of Viruses that Interact with the Host Innate Immune System
- Ary, Beatrice Elisabeth
- Advisor(s): Craik, Charles S
Abstract
Cells have evolved proteins that detect foreign DNA, RNA, or proteins which then activate cellular pathways to combat bacterial or viral infection.1–4 Apolipoprotein B editing complex 3 (APOBEC3 or A3) is a host cytidine deaminase that deaminates cytidine to uridine in viral DNA in the cytoplasm causing hyper G→A mutation leading to destabilization and degradation of the viral genome.5–8 HIV viral infectivity factor (vif) has evolved to regulate A3 degradation of viral DNA.9 Vif hijacks host ubiquitination machinery to degrade A3s and prevent packaging of A3 into the viral particle.10,11 Vif hijacks the cotranscription factor core factor binding unit beta (CBF-beta), Elongin B (ELOB) and Elongin C (ELOC) to form the VCBC complex.12–15 VCBC binds A3s and Cullin 5 (Cul5) for ubiquitination and degradation of A3 thereby preventing packaging.12,16 Antigen binding fragments (Fabs) were generated against VCBC using a naïve B-cell Fab phage display library to isolate biological tools that are specific for the host-virus interaction.17 Two high affinity Fabs were found to bind at distinct epitopes on VCBC and produced distinct phenotypes when expressed in cells as single chain variable fragments (scFvs).17 The Fab 3C9 shields A3F from ubiquitination and restores packaging of A3F into the viral particle. The Fab 1D1 blocks binding of Cul5 and ubiquitination in vitro. The scFv 1D1 prevented ubiquitination of A3F but did not restore packaging. Affinity purification mass spectroscopy (AP-MS) in HEK293Ts with 3C9 scFv and 1D1 scFv showed different interactomes in the presence of vif. AP-MS with 1D1 scFv did not interact with CBF-beta, an important component of the VCBC complex. Spatial and temporal elucidation of proteins interacting with the complex will further determine events effecting viral infectivity.
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