Student Theses and Dissertations
Visualizing Protease Activation, Retrochmp3 Activity, and VPR Recruitment During HIV-1 Assembly
Date of Award
Doctor of Philosophy (PhD)
This thesis covers my contributions to the field of HIV-1 assembly and the field of science communication. Over the course of my studies, I have determined when the HIV-1 protease becomes active during the assembly of new virions, elucidated the mechanism by which the protein retroCHMP3 has an antiviral function, and examined the kinetics of recruitment of the HIV-1 accessory protein Vpr. I have also added significantly to science communication research through a study which evaluated the effectiveness of science summary methods. HIV-1 virions assemble at the plasma membrane of infected cells. The assembly of new viruses is driven by the HIV-1 Gag structural polyprotein and involves a number of viral and host proteins to produce infectious virions. Although many of these factors have been studied extensively, there are still steps during viral assembly that have yet to be fully characterized. In Chapter 2 of this thesis, I will discuss my study of the activation of the HIV-1 protease which is necessary for infectious particle production. Previous research has suggested that protease becomes active prior to scission of the particle from the cell, but there was no study which specifically measured protease activation and its cleavage of Gag during the assembly process. The timing of protease activation and Gag cleavage directly affects particle morphology and infectivity. Using polarization TIRF microscopy, I have directly measured protease activation and Gag cleavage. My results suggest that protease becomes active prior to recruitment of the host ESCRT proteins and scission from the cell. Chapter 3 of this thesis describes my work on the protein retroCHMP3, which was performed in collaboration with Sundquist lab at the University of Utah. The retroCHMP3 protein was originally found in squirrel monkeys and is a truncated and mutated version of the endogenous CHMP3 ESCRT protein. Data from the Sundquist lab shows that retroCHMP3 inhibits the budding of many ESCRT-dependent viruses, including HIV-1, while not preventing essential ESCRT-dependent cell functions such as cytokinesis. To determine the mechanism of retroCHMP3, I used TIRF imaging to show that retroCHMP3 alters ESCRT recruitment to sites of HIV-1 assembly. This altered recruitment suggests that a delay in scission occurs when retroCHMP3 is present in cells. My results also suggest that there is a loss of HIV-1 proteins back into the cell due to protease activation as assayed by loss of fluorescence. As a confirmation of these results, I show that this loss of fluorescence was rescued by genetic inactivation of the protease. We suspect that the retroCHMP3 alteration of ESCRT function affects viral budding more than cellular events like cytokinesis due to the need for viruses to assemble quickly to avoid cellular defense mechanisms and loss of viral proteins from protease activation. My final contribution to the HIV-1 field is a study of the recruitment kinetics of the HIV-1 accessory protein Vpr. My study of Vpr will also be presented in Chapter 3 of this thesis. Vpr is a protein which increases infectivity in infected animals and humans. It is packaged specifically into virions through interactions with the Gag protein. Through simultaneous TIRF imaging of Gag and Vpr, I was able to show that Vpr has a delayed accumulation compared to Gag, suggesting that Vpr is not bound to Gag before coming to the membrane. This work hints at possible interactions between Vpr and the host ESCRT proteins which have recruitment sites close to the Vpr recruitment site. Finally, Chapter 4 of this thesis will show my findings regarding the effectiveness of different science summaries. Science summaries like video abstracts, graphical abstracts, and plain language summaries all help with accessibility of research papers. To study the efficacy of each of those summaries, I created a survey which showed participants video abstracts, graphical abstracts, plain language summaries, and academic abstracts from two HIV-1 research papers. My findings suggested that video abstracts and plain language summaries are both effective ways to summarize scientific research while graphical abstracts and academic abstracts are not as effective. After presenting all of my work in Chapters 2-4, I will discuss the implications and immediate future directions of each contribution I have made. These discussions will be in Chapter 5 of this thesis.
Bredbenner, Kate, "Visualizing Protease Activation, Retrochmp3 Activity, and VPR Recruitment During HIV-1 Assembly" (2020). Student Theses and Dissertations. 694.
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy