Biochemical and biophysical investigations of HIV-1 viral infectivity factor and its interaction with human APOBEC3G
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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 the APOBEC3G and the E3 ubiquitin ligase complex. Human APOBEC3G is a potent inhibitor of retroviruses and functions to deaminate cytidines to uridines in HIV-1 ssDNA during viral replication. Thus, degradation of APOBEC3G neutralized one of the body’s main defenses against HIV. The characterization of the Vif - APOBEC3G interaction is important to understand how Vif functions in suppressing APOBEC3G activity, which could lead to new and better HIV drugs and therapies. However, the study of the Vif - APOBEC3G complex has been severely hindered by the fact that both Vif and APOBEC3G proteins have proved to be very difficult to express and purify in large amounts. The goal of the work presented in this dissertation is twofold: (1) to address the current bottleneck for Vif and APOBEC3G protein production by developing expression and purification protocols that are capable of producing milligram quantities of both proteins and (2) characterize the Vif protein and the Vif-APOBEC3G complex using biochemical and biophysical methods. Milligram quantities of Vif were produced using the bacterial expression system. The protein has been characterized using a number of biochemical/biophysical techniques and suggest that Vif is an unstructured protein under native conditions. Circular dichroism (CD) indicated a random coil structure with few secondary structural elements. Nuclear magnetic resonance (NMR) produced a spectrum characteristic of an unstructured protein. Small-angle Xray scattering (SAXS) studies suggested that Vif forms multimers with a compact core in solution. Overall, these results support the idea that Vif is an intrinsically unstructured protein in solution. The full-length APOBEC3G was produced using a baculovirus expression system. Both monomeric and dimeric APOBEC3G forms were observed providing the first direct evidence for APOBEC3G dimerization. The binding kinetics of Vif-APOBEC3G interaction was determined by surface plasmon resonance (Biacore). Interestingly, full-length Vif was observed to interact with APOBEC3G dimers (KD=0.2 nM) with an affinity that is 1000-fold greater than that observed for the APOBEC3G monomers (KD=200 nM).