Date of Award

2021

Document Type

Thesis

Degree Name

M.S.

Department

Biological Sciences

First Advisor

Schuyler Van Engelenburg

Second Advisor

Michelle Knowles

Third Advisor

Erich Kushner

Fourth Advisor

Cedric Asensio

Keywords

HIV-1, Superresolution microscopy

Abstract

In my findings, I have established a set series of protocols to recombinantly produce, purify and apply various fluorescent probes in vitro for the fluorescent labeling and study of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein during HIV viral assembly. There remains insufficient knowledge about the molecular dynamics and interactions of HIV-1 Env protein with its counterpart, Gag, on the inner host cell surface during assembly of a mature virus particle. There also remains an insufficient amount of data for the understanding and clarification of the mechanism of action of a known host cell HIV-1 restriction factor, Serinc5 (SER 5), which is believed to have correlation with Env in fusion events.

Using superresolution microscopy to measure and track host–pathogen interfaces that occur on a scale below the resolution limit of the light microscope is becoming increasingly popular. I have used total internal reflection microscopy (TIRF) and Photoactivated Localization Microscopy (PALM) to depict sites of molecular assembly via Env and Gag colocalization as well as measure and track other means of Env such as diffusion rates and host cell restriction factor interactions.

Overall, my findings have provided improved strategies and tools for the use in fluorescently labeling and tracking HIV-1 Env and an HIV-1 host cell restriction factor known as SER 5. Superresolution microscopy and single particle tracking can be achieved provided high specificity, brightness and purity while maintaining low KDs. The production and data of these probes are imperative for both understanding the pathological processes of HIV-1 assembly and other stages of the HIV-1 life cycle as well as the various protein-protein interactions.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Provenance

Received from ProQuest

Rights holder

Merissa Michelle Bruns

File size

82 pgs

File format

application/pdf

Language

en

Discipline

Biophysics, Virology, Immunology

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