Human immunodeficiency virus (HIV) targets and depletes CD4+ T cells, compromising the body's ability to fight off infections. Progressed HIV disease can lead to impaired renal function known as HIV-associated nephropathy (HIVAN). Epithelial cells are typically ineffective targets of HIV-1 as they lack the CD4 and CCR5 molecules that are involved in viral entry into CD4+ T cells. However, previous research in the laboratory of Dr. Benjamin K. Chen demonstrated that renal tubule epithelial (RTE) cells were capable of viral uptake through a T cell-mediated, but CD4-independent mechanism. In addition, experiments implicated heparan sulfate proteoglycans (HSPG) as possible attachment receptors for HIV-1 through their heparan sulfate (HS) polysaccharide chains. The addition of anti-HSPG and anti-syndecan 1 antibodies blocked virus transfer by approximately 50%, suggesting a role for HSPG in viral entry. As a result, the syndecan (SDC) class of heparan sulfate receptors were assessed for their potential to serve as attachment receptors for HIV-1 through knockout studies. A co-culture system with donor HIV-expressing Jurkat T cells and target renal tubular epithelial (HK2) cells were used as a system for HIVAN pathogenesis and enabled cell-to-cell viral transfer. Despite the generation of stable SDC gene knockout lines, no change in viral transfer was observed, suggesting redundant or alternate pathways for HIV-1 entry. Understanding viral entry into epithelial cells is crucial as these sites can serve as reservoirs for HIV-1, where it can continue to replicate even when plasma viral load has been sufficiently reduced with antiretroviral treatment.

<p>Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy. </p><p>Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.</p> / Dissertation