INTERACTION OF ONCORETROVIRAL GAG PROTEIN WITH HOST NUCLEAR FACTORS
Open Access
- Author:
- Rice, Breanna Lynn
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 05, 2018
- Committee Members:
- Leslie Joan Parent, Dissertation Advisor/Co-Advisor
Sarah Bronson, Committee Chair/Co-Chair
Rebecca C Craven, Committee Member
Sergei A Grigoryev, Committee Member
Kristin Ann Eckert, Outside Member - Keywords:
- Retrovirus
Rous Sarcoma Virus
Virology
HIV
Nucleus - Abstract:
- Retroviruses are obligate parasites that cause cancer and immunodeficiences in humans and animals. Their RNA genomes undergo reverse transcription into a DNA intermediate that is integrated into the host genome. Following integration, the virus requires use of the host cell’s transcription machinery to produce the mRNAs needed for protein translation as well as the unspliced transcript that serves as the packaged genome. The structural protein of retroviruses, Gag, is responsible for binding the genome and packaging it into virions. The initial interaction between Gag and the genome was always thought to occur in the cytoplasm where the mRNA is translated into the Gag protein. However, pioneering work in our laboratory has demonstrated the Gag protein from the Rous sarcoma virus (RSV) undergoes nuclear trafficking, which is necessary for efficient genomic RNA packaging. RSV Gag utilizes the nuclear import proteins Importin α/β, Importin 11, and TNPO3 for nuclear entry, and the CRM1 pathway for nuclear egress. Since our discovery of the functional significance for Gag nuclear trafficking, the nuclear trafficking of other retroviral Gag proteins has begun to be more extensively studied. RSV Gag forms discrete foci within the nucleus, and when Gag is trapped in the nucleus by blocking the CRM1-dependent export, the number of foci significantly increases. These foci exhibit characteristics that are similar to host nuclear bodies. Gag proteins move in and out of the foci rapidly, with a half-time of recovery of approximately 8 seconds. The overall goal of the research performed for this dissertation is to characterize the nuclear foci of RSV Gag and determine what host protein(s) could be functioning as the tether for these foci. We also wanted to determine whether there are host factors that Gag may use within the nucleus to locate the genomic viral RNA. In this dissertation, I will be discussing my work that demonstrates novel interactions Gag may have with cellular nuclear factors. Findings presented in this dissertation demonstrate that the RSV Gag nuclear foci display obstructed diffusion, indicating the foci are tethered to a protein or RNA. To determine where in the nucleus Gag localizes, fractionation experiments were performed that showed RSV Gag, as well as human immunodeficiency virus (HIV) Gag were able to be extracted from euchromatin and heterochromatin protein fractions. To uncover host nuclear proteins that Gag could be interacting with, mass spectrometry experiments were performed and it was found that RSV and HIV-1 Gag proteins may interact with transcription-related proteins, splicing factors, and chromatin remodeling proteins. Colocalization experiments indicate that RSV Gag colocalizes with splicing factors SC35 and SF2 to a high degree, and that when the expression of SC35 is increased in cells, the number of RSV Gag foci increase. We found that when expression of the nuclear import protein, TNPO3 is increased, the amount of nuclear Gag increases. This is interesting because of the role TNPO3 has in transporting splicing factors to splicing speckles, suggesting Gag may use TNPO3 to localize to splicing speckles. Unpublished lab results show that the RSV and HIV Gag proteins associate with the viral RNA in the nucleus. While it has been published that certain Gag proteins or domains are able to interact with chromatin during integration, the work presented here, along with our unpublished results lead us to hypothesize that Gag may be travelling to sites of transcription through interactions with cellular factors, which allows Gag to bind the viral RNA co-transcriptionally for packaging. This is a novel model that challenges the dogma of where retroviruses package their genome, and presents new roles for Gag in the nucleus.