Development of Imaging Techniques for HIV-1 Cell-Cell Contacts and T-cells

Abstract

Human Immunodeficiency Virus 1 (HIV-1) became a global pandemic in the early 1980s, continuing to affect millions of people every year. Though progress has been made in the last four decades, there are no FDA approved therapeutic classes for the assembly stage in the HIV-1 replication cycle. Previous studies have found the HIV-1 can become drug resistant by increasing its prevalence to infect naïve cells by cell-cell contact. Therefore, it is more important now than ever to study the spread of HIV-1 from cell-cell contacts. First, we establish a robust cell-cell contact imaging assay with T-cells and monocyte-like macrophage (MLM). We, then, combine this pulse-chase assays to investigate the mobility of internalized Env to sites of cell-cell contact and determine that exocytosis to these sites was not seen on a large or readily rapid rate. Next, we combined our established cell-cell contact system with lithography masks and hydrophobic compound Lipidure to only allow cells to lay down on areas of the imaging coverslip that were ablated. This was used for adherent immune cells as well as improved upon to establish an imaging method for suspension T-cells. We found an increase in the number of cell-cell contacts with our method. Following this, we use single channel superresolution microscopy to quantify the relative monomeric and dimeric CD4 population. Here, we provided an orthogonal approach to the biochemical verification for the chimera’s monomeric nature. Lastly, we use simultaneous, dual-color superresolution imaging to investigate the nanometer organization of viral proteins and receptors at sites of cell-cell contact.