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Randi Mackler Dissertation FINAL.pdf (6.15 MB)

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Understanding Prototype Foamy Virus Integrase Site Selection, Activity, and Stability

Mackler, Randi Michelle
http://orcid.org/0000-0002-2513-6479
http://rave.ohiolink.edu/etdc/view?acc_num=osu1542306356468134

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Biomedical Sciences.
HIV is a worldwide pandemic that remains incurable. Recent statistics show that in the United States alone, ~15 per 100,000 people were newly infected with HIV-1 in one year. The barrier to a cure is a reservoir of cells with viral DNA stably integrated into their genome, yet are not killed by the immune system. The integration step of the retroviral life cycle is crucial in reservoir formation. Viral DNA integration is catalyzed by protein integrase (IN). We study HIV-1 IN and prototype foamy virus (PFV) IN. PFV IN is used to model for HIV-1 integration, as HIV-1 IN inhibitors also block PFV IN activity. This implies that the two proteins have similar catalytic mechanisms. However, we have found differences between PFV IN and HIV-1 IN function. We determined that PFV IN could utilize calcium for strand transfer, unlike HIV-1 IN. Additionally, though HIV-1 IN has been reported to rapidly commit to its target DNA, PFV IN does not commit within an hour. There are likely differences in searching and target capture mechanisms between the two INs. A benefit to using PFV IN is that it can be readily assembled with oligomers that mimic viral cDNA ends to form an intasome complex. The PFV intasome contains a tetramer of PFV IN and two oligomer DNAs. We found in vitro that these intasomes aggregate at 37°C. Full-length intasomes aggregate more than those containing truncated PFV IN outer subunits, particularly deleting the carboxyl terminal domain (CTD). Aggregation can be prevented by using high non-physiological salt concentrations or with addition of small molecule protocatechuic acid (PCA). This finding is useful for future experiments that require longer lifetimes of PFV intasomes. Integration into chromatin is not well understood. Chromatin is comprised of basic units called nucleosomes. Our goal is to understand how IN chooses its site when integrating into nucleosomes. We altered either nucleosomes or PFV IN to understand how changes impact integration activity and site specificity. Perturbations include using nucleosomes with specific histone posttranslational modifications (PTMs), altering salt concentration, and utilizing PFV IN truncation mutants. We hypothesize major grooves with highly bent DNA not occluded by histone proteins are the most favored sites. However, our studies showed that not every distorted and exposed major groove is favored, suggesting that there is more to PFV IN site selection. Deleting the CTDs of the outer subunits of the IN tetramer greatly increases integration efficiency to linear DNA, but this effect is largely lost with integration into nucleosomes. Truncated mutant complexes are the most impacted by increasing salt concentrations, and affinity purification experiments showed that PFV IN ¿CTD intasomes interact weakly with nucleosomes compared to wild type. Our data supports the hypothesis that CTDs of PFV IN directly bind to nucleosomes. The interaction is at least partially mediated by histone tails. These data will expand knowledge in the field and will be crucial to development of novel therapeutics to combat HIV-1. This work can also inform design of novel retroviral gene therapy vectors.
Kristine Yoder (Advisor)
Michael Freitas (Committee Member)
Jesse Kwiek (Committee Member)
Li Wu (Committee Member)
224 p.
Biochemistry; Biology; Biomedical Research; Virology
integrase; prototype foamy virus; nucleosome; biochemistry; retrovirus; virus; HIV-1; gene therapy

Recommended Citations

Citations

  • Mackler, R. M. (2018). Understanding Prototype Foamy Virus Integrase Site Selection, Activity, and Stability [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1542306356468134

    APA Style (7th edition)

  • Mackler, Randi. Understanding Prototype Foamy Virus Integrase Site Selection, Activity, and Stability. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1542306356468134.

    MLA Style (8th edition)

  • Mackler, Randi. "Understanding Prototype Foamy Virus Integrase Site Selection, Activity, and Stability." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1542306356468134

    Chicago Manual of Style (17th edition)

osu1542306356468134
454
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This open access ETD is published by The Ohio State University and OhioLINK.