Spatiotemporal properties of human immunodeficiency virus type 1 Gag protein and its interaction with host transcription machinery

Restricted (Penn State Only)
- Author:
- Chang, Jordan
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 29, 2024
- Committee Members:
- Feng Yue, Special Member
Lisa Shantz, Program Head/Chair
Nicholas Buchkovich, Major Field Member
Leslie Parent, Chair & Dissertation Advisor
Zhonghua Gao, Outside Unit & Field Member
Clare Sample, Major Field Member - Keywords:
- HIV-1
Gag
nuclear trafficking
confocal microscopy
live cell imaging
euchromatin
transcription - Abstract:
- Retroviruses are single-stranded RNA viruses that can cause a variety of cancers and diseases, including leukemia, sarcomas, and acquired immunodeficiency syndrome (AIDS). One hallmark of retroviruses is their ability to integrate into the host chromosome and hijack host transcription and splicing machinery to promote its viral expression. The most notable human retrovirus is the human immunodeficiency virus type-1 (HIV-1) which leads to the development and AIDS, rendering infected individuals to succumb to opportunistic infections. HIV-1 continues to be a global pandemic since its discovery in the 1980s. Upon infection into host T cells, the HIV-1 viral genome undergoes reverse transcription to generate DNA-intermediate that is integrated into the host chromosome. From here, HIV-1 recruits RNA polymerase II and various transcription factors to produce the viral mRNA needed to express other viral accessory proteins, the structural protein Gag, and Gag-Pol. The main function of Gag is to select and bind to the unspliced viral RNA for packaging during viral assembly. It was previously thought that the initial interaction between Gag and its packaged viral RNA occurs in the cytoplasm. However, work from our laboratory has demonstrated that HIV-1 Gag, and other retroviral Gag proteins can undergo nuclear localization. This finding raises the possibility that HIV-1 genome packaging begins within the nucleus of the cell. Previous work studying HIV-1 Gag demonstrated that Gag is able to associate with the unspliced viral RNA in the nucleus. Moreover, Gag forms discrete foci in the cell when observing it under confocal microscopy. Since this initial finding, HIV-1 Gag has been shown to interact with a variety of host nuclear factors involved in RNA processing, transcription, and chromatin remodeling. Furthermore, Gag and viral RNA co-localization increased upon treatment with transcription inhibitor actinomycin D. These findings led us to wonder what the role nuclear HIV-1 Gag is and how does Gag nuclear trafficking occur. In this study, we sought to better understand the dynamic properties of HIV-1 Gag by addressing what, when, where, and how. What does nuclear HIV-1 Gag interact with? When does Gag nuclear trafficking begin? Where within the nucleus does HIV-1 Gag localize? How does nuclear trafficking occur? Using a variety of biochemical and imaging techniques, we were able to visualize the movement of HIV-1 Gag in vivo using live cell microscopy. Furthermore, we began to decipher how the different Gag protein domains contribute to Gag nuclear localization. We also found that a subset of HIV-1 Gag begins trafficking to the nucleus shortly after expression and preferentially localizes to transcriptionally active euchromatin regions. Lastly, we demonstrate that HIV-1 Gag can interact with host RNA polymerase II. Taken together, results from this study led us to propose a model in which HIV-1 Gag is localizing with RNA polymerase II at active viral transcription sites to select for the unspliced viral RNA co-transcriptionally.