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Visualization of the Intracellular HIV-1 Replication Cycle

Abstract Details

2017, Doctor of Philosophy, Case Western Reserve University, Molecular Virology.
The sequence of events from HIV-1 entry through transcription of proviral DNA is not well understood. While many studies have attempted to investigate these post-fusion events, the unstable nature of intracellular HIV-1 particles has made biochemical analyses difficult. Developments in imaging techniques have given a glimpse of intracellular HIV-1 particles during active infection, but few of these attempts have connected both the early and late events of HIV-1 infection in primary human target cells. Even fewer of these attempts effectively visualized actively transcribing viruses that contribute to productive infection. Here, we developed a method to visualize HIV DNA reverse transcription (RT) products by incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU) in infected monocyte-derived macrophages (MDM). We observed distinct EdU puncta in the cytoplasm and nuclei of infected cells which increased in number following depletion of the dNTPase SAMHD1. Interestingly, we found HIV-1 capsid associated with HIV-1 DNA in the cytoplasm and nuclei of infected MDM. Our assay also represented improvements upon current qPCR techniques in measuring the efficiency of nuclear import of two CA mutants, N74D and Q63A/67A, and showed marked sensitivity in detecting intranuclear HIV-1 genomes in productively and non-productively infected MDM. Because we were able to track nuclear HIV-1 DNA through productive infection of MDM, we were interested in whether we could identify actively transcribing proviruses. Using RNA fluorescence in situ hybridization (FISH) we were for the first time able to image actively transcribing proviruses through colocalization of both HIV-1 DNA and HIV-1 RNA. These transcribing proviruses were further confirmed through colocalization of nuclear HIV-1 DNA and RNA with specific HIV-1 transcription factors. Modification of our newly developed RNA FISH technique to monitor HIV-1 transcription in productively infected and reactivated, HIV-latently infected CD4 T cells identified specific intranuclear foci of HIV-1 RNAs. These foci correlated with the number of proviral integration sites in latently infected cell lines, suggesting RNA FISH detects sites of proviral transcription in these cells. Overall, our novel HIV-1 imaging techniques allow for stable labeling and monitoring of functional HIV-1 complexes within infected cells during cytoplasmic transit, nuclear import, and transcription.
David McDonald (Advisor)
John Tilton (Committee Chair)
Jacek Skowronski (Committee Member)
George Dubyak (Committee Member)
167 p.

Recommended Citations

Citations

  • Stultz, R. D. (2017). Visualization of the Intracellular HIV-1 Replication Cycle [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1496328611715148

    APA Style (7th edition)

  • Stultz, Ryan. Visualization of the Intracellular HIV-1 Replication Cycle. 2017. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1496328611715148.

    MLA Style (8th edition)

  • Stultz, Ryan. "Visualization of the Intracellular HIV-1 Replication Cycle." Doctoral dissertation, Case Western Reserve University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1496328611715148

    Chicago Manual of Style (17th edition)