Molecular Determinants of CCR5-Tropic Human Immunodeficiency Virus 1 Entry Into Low-CCR5 Expressing Cells

Abstract

Over 80 million people have been infected with Human Immunodeficiency Virus 1 (HIV-1) worldwide. Despite advances in the treatment of HIV-1 infected individuals, practical cure or prevention of HIV-1 infections has not been achieved. Basic research on HIV-1 infection is imperative to better understand and prevent new infections. It is currently known that the HIV-1 envelope glycoprotein (Env) binds immune cell receptors CD4 and CCR5, sequentially. Binding to each receptor triggers conformational changes in Env that promote viral fusion and entry. Researchers seek to gain insight into the CCR5-induced transition from the Env pre-hairpin intermediate to downstream fusion-active conformations, as CCR5 plays an essential role in infection. The structures assumed by Env after CCR5 binding and before membrane fusion, in which the protein assumes a six-helix bundle conformation, remain unknown. In my work, I tested the essential role of CCR5 by attempting to adapt HIV-1 to infect cells with reduced levels of CCR5. To do so, I generated cells with titratable cell surface CCR5 expression levels using a tet-promotor system, and passaged virus in these cells in the presence of doxycycline. In doing so, I obtained an HIV-1 capable of infecting cells expressing ~1300 CCR5 molecules/cell. Changes in Env mapped to gp120 and gp41, the surface and transmembrane subunits of Env. These changes conferred either enhanced replication capacity or allowed Env to expose receptor-binding sites in the absence of receptor. Functional analysis of the adapted HIV-1 helped to identify new regions in Env critical to CCR5-mediated entry kinetics and viral fusion. This work will also help us to understand the pathways of HIV-1 escape from CCR5 inhibitors and provide information about the vulnerability of Env intermediate conformational states to inhibition by drugs and antibodies. New tools to handle CCR5 for biochemical studies could help researchers isolate these intermediate conformational states. I therefore generated lipid bilayer nanodiscs containing human CCR5 molecules derived from mammalian CHO cells. The reconstituted CCR5 maintained its capacity to bind the anti-CCR5 monoclonal antibody 2D7 and HIV-1 gp120. Optimization of CCR5 nanodiscs can allow this tool to be used in future studies of CCR5-bound HIV-1.