Molecular Characterization of the EWI-2 Fusion Inhibitory Mechanism in the Context of HIV Infection

Abstract

Human Immunodeficiency Virus (HIV) can be transmitted cell-to-cell through the formation of a virological synapse (VS). The VS is a contact between an infected cell and an uninfected T cell (target), formed by binding between the viral envelope glycoprotein (Env) on the surface of an infected cell and the receptor for HIV (CD4) on target cells. Although it might seem likely that interaction between producer and target cells would often result in cell-cell fusion, the VS primarily resolves in complete cell separation. Cell-cell fusion at the VS is prevented by both viral and host fusion inhibitory mechanisms. Fusion inhibitory host proteins include EWI-2. Although EWI-2 is a known fusion inhibitor, this protein’s mechanism of fusion inhibition is not understood. The goal of this project was to characterize the EWI-2 fusion inhibitory mechanism. This project determined optimal transfection conditions for EWI-2 mutant plasmids to obtain equal surface expression of each EWI-2 construct using fluorescence microscopy. This project also designed a new fusion assay system, using nanoluciferase as a reporter, which will be used for future fusion assays to characterize EWI-2’s fusion inhibitory mechanism. We hypothesize that the EWI-2 extracellular domains will play a major role in EWI-2 mediated fusion inhibition.

Primary Faculty Mentor Name

Markus Thali

Graduate Student Mentors

Emily Whitaker

Faculty/Staff Collaborators

Emily Whitaker (Graduate Student Mentor)

Status

Undergraduate

Student College

College of Agriculture and Life Sciences

Program/Major

Microbiology and Molecular Genetics

Primary Research Category

Biological Sciences

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Molecular Characterization of the EWI-2 Fusion Inhibitory Mechanism in the Context of HIV Infection

Human Immunodeficiency Virus (HIV) can be transmitted cell-to-cell through the formation of a virological synapse (VS). The VS is a contact between an infected cell and an uninfected T cell (target), formed by binding between the viral envelope glycoprotein (Env) on the surface of an infected cell and the receptor for HIV (CD4) on target cells. Although it might seem likely that interaction between producer and target cells would often result in cell-cell fusion, the VS primarily resolves in complete cell separation. Cell-cell fusion at the VS is prevented by both viral and host fusion inhibitory mechanisms. Fusion inhibitory host proteins include EWI-2. Although EWI-2 is a known fusion inhibitor, this protein’s mechanism of fusion inhibition is not understood. The goal of this project was to characterize the EWI-2 fusion inhibitory mechanism. This project determined optimal transfection conditions for EWI-2 mutant plasmids to obtain equal surface expression of each EWI-2 construct using fluorescence microscopy. This project also designed a new fusion assay system, using nanoluciferase as a reporter, which will be used for future fusion assays to characterize EWI-2’s fusion inhibitory mechanism. We hypothesize that the EWI-2 extracellular domains will play a major role in EWI-2 mediated fusion inhibition.