Files
Abstract
The human immunodeficiency virus type-1 (HIV-1) Virus Infectivity Factor (Vif)mediates the degradation of a cellular antiviral factor, Apolipoprotein B mRNA-editing enzymecatalytic,polypeptide-like 3G (APOBEC3G), by serving as an adaptor that bridges theAPOBEC3G and the E3 ubiquitin ligase complex. Human APOBEC3G is a potent inhibitor ofretroviruses and functions to deaminate cytidines to uridines in HIV-1 ssDNA during viralreplication. Thus, degradation of APOBEC3G neutralized one of the bodys main defensesagainst HIV. The characterization of the Vif - APOBEC3G interaction is important to understandhow Vif functions in suppressing APOBEC3G activity, which could lead to new and better HIVdrugs and therapies. However, the study of the Vif - APOBEC3G complex has been severelyhindered by the fact that both Vif and APOBEC3G proteins have proved to be very difficult toexpress and purify in large amounts.The goal of the work presented in this dissertation is twofold: (1) to address the currentbottleneck for Vif and APOBEC3G protein production by developing expression andpurification protocols that are capable of producing milligram quantities of both proteins and (2)characterize the Vif protein and the Vif-APOBEC3G complex using biochemical andbiophysical methods.Milligram quantities of Vif were produced using the bacterial expression system. Theprotein has been characterized using a number of biochemical/biophysical techniques andsuggest that Vif is an unstructured protein under native conditions. Circular dichroism (CD)indicated a random coil structure with few secondary structural elements. Nuclear magneticresonance (NMR) produced a spectrum characteristic of an unstructured protein. Small-angle Xrayscattering (SAXS) studies suggested that Vif forms multimers with a compact core insolution. Overall, these results support the idea that Vif is an intrinsically unstructured protein insolution.The full-length APOBEC3G was produced using a baculovirus expression system. Bothmonomeric and dimeric APOBEC3G forms were observed providing the first direct evidence forAPOBEC3G dimerization. The binding kinetics of Vif-APOBEC3G interaction was determinedby surface plasmon resonance (Biacore). Interestingly, full-length Vif was observed to interactwith APOBEC3G dimers (KD=0.2 nM) with an affinity that is 1000-fold greater than thatobserved for the APOBEC3G monomers (KD=200 nM).