Thermodynamic Analysis and Characterization of Viral-Host Protein Interactions as a Preface to HIV Therapeutic Design

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2013.The infection and propagation of the human immunodeficiency virus type 1 (HIV- 1) within a host depends on successful interactions between viral and cellular proteins. In some cases the virus has evolved pathways to hijack the cellular machinery in order to evade the innate immune factors that would otherwise restrict viral infectivity. For example, the HIV-1 auxiliary protein Vif plays an essential role in host-cell infection by suppressing the activity of the host’s innate antiretroviral proteins in the APOBEC3 (A3) family. The mechanism of action for Vif entails binding to A3 proteins in order to recruit them to the host’s own Cullin-RING E3 ubiquitin ligase (CRL), resulting in their ubiquitination and degradation. The CRL comprises the cellular proteins Cullin5 (Cul5), ElonginB, ElonginC (EloB/C), and Rbx2. Recently the human protein CBFβ was found to be essential for HIV-1 infectivity and it is indispensible for Vif recruitment of A3 proteins to the CRL. At present, the role of CBFβ within the CRL remains elusive. To better define the binding affinity and interfaces of various proteins present in Vif-hijacked CRL complexes I developed methods for bacterial expression and purification of: (i) CRL-like complexes comprising HIV-1 Vif/CBFβ/EloB/C, (ii) human Cul5, (iii) the Vif/CBFβ/EloB/C/Cul5 complex, and (iv) human A3C. Using isothermal titration calorimetry (ITC) I found that CBF increases the relative affinity of Cul5 binding to CRL-like complexes by 60-fold compared to CRL-like complexes in which N-terminally truncated Vif(95-192) was utilized to eliminate CBF binding. Heat capacity measurements were negative (ΔCP = -0.52) and correlated the binding of Cul5 to CRL-like complexes to a large burial of hydrophobic residues. In order to identify specific regions of HIV-1 Vif that interact with Cul5 in CRL-like complexes, the following samples were subjected to hydrogen/deuterium exchange mass spectrometry: Vif/CBFβ/EloB/C, Vif/CBFβ/EloB/C/Cul5, and Cul5. The results revealed a significant number of regions within Vif that become protected upon Cul5 binding including novel regions that have not been described in the literature. Collectively my work provides a global map for protein interactions within the pentamer complex that provides a basis for rational experimental analysis of viral-host protein interactions