Determinants of HIV-1 Gag Localization to Uropods in Polarized T Cells and the Role Uropods Play in Virus Spread.

Abstract

HIV-1 is a deadly virus that has killed millions of people around the world. One of the primary targets of HIV-1 in the human body is T cells. In lymphoid organs, such as lymph nodes, T cells are highly motile and adopt a polarized morphology. However, little is known about how HIV-1 localizes, assembles or spreads in polarized T cells. Thus, determining the molecular mechanisms utilized by HIV-1 in polarized T cells can help to understand the virus in vivo. Polarized T cells are characterized by a leading edge at the front and a rear end protrusion called a uropod. In this thesis, it was determined that the HIV-1 structural protein Gag localizes to the uropod in polarized T cells. HIV-1-expressing T cells were found to contact uninfected target cells preferentially via uropods relative to leading edges. Also, uropods participated in virological synapses that mediate HIV-1 cell-to-cell transmission. Together, these findings indicate that uropods play an important role in HIV-1 spread in vivo. Nucleocapsid (NC) is a structural domain of Gag that promotes Gag multimerization by using RNA as a scaffold. NC-mediated multimerization, at the level of tetramerization or higher, was found to be required for uropod localization of Gag. Gag was found to copatch with some (CD43, PSGL-1, CD44), but not all (ICAM-1, ICAM-3, CD59) uropod markers, even in unpolarized T cells. These data, along with live cell analyses, indicate that Gag associates with a specific subset of uropod-directed microdomains (UDMs) that could carry Gag to the uropod. The specificity of Gag association with UDMs was found to be determined by matrix (MA), another Gag structural domain responsible for plasma membrane binding. The observations made in this thesis suggest a working model of HIV-1 replication and spread in polarized T cells: When NC-mediated multimerizaton reaches the level of tetramerization or higher, Gag associates with specific uropod-directed microdomains enriched in CD43, PSGL-1 and CD44 via an MA-mediated mechanism. These Gag-associated UDMs then laterally localize on the membrane to the uropod where virus particles assemble and accumulate. Uropods then mediate contact and VS formation with target cells to facilitate cell-to-cell transmission.