University Library
  • Login
A gateway to Melbourne's research publications
Minerva Access is the University's Institutional Repository. It aims to collect, preserve, and showcase the intellectual output of staff and students of the University of Melbourne for a global audience.
View Item 
  • Minerva Access
  • Medicine, Dentistry & Health Sciences
  • Melbourne Medical School
  • Microbiology & Immunology
  • Microbiology & Immunology - Theses
  • View Item
  • Minerva Access
  • Medicine, Dentistry & Health Sciences
  • Melbourne Medical School
  • Microbiology & Immunology
  • Microbiology & Immunology - Theses
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

    Following the HIV-1 RNA footprint in cells with latent provirus: reversing silent infection through Tat

    Thumbnail
    Download
    Complete Thesis (90.31Mb)

    Citations
    Altmetric
    Author
    Lee, Michelle
    Date
    2018
    Affiliation
    Microbiology & Immunology
    Metadata
    Show full item record
    Document Type
    PhD thesis
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/216475
    Description

    © 2018 Dr Michelle Lee

    Abstract
    Globally, the HIV-1 epidemic remains robust and the size of the infected population continues to grow, particularly in sub-Saharan Africa. Although viral suppression is achieved through administration of cART, therapy is lifelong. A compartment of cells that carry HIV-1 in a transcriptionally inactive state, but which retains replicative potential, persists in infected individuals and re-emerges to seed infection when treatment is interrupted. Use of latency reversing agents for perturbation of this reservoir has been shown to be ineffective in the clinical context. This stresses the need for the development of more refined approaches to reactivate latent infection. Multiple layers of repression are present in the cell and at the latent HIV-1 promoter. A central aspect of HIV replication that is blocked during latency is the process of transcriptional elongation. In productive infection, this stage of transcription is enhanced by the action of the viral protein, Tat. Hence, reactivation of latency may be possible through inducing the expression of Tat in a latently infected cell. From the pre-existing DNA template in cells with silenced proviral genomes, tat sequences can be transcribed by a process independent of the 5’ LTR. Generation of readthrough transcripts containing sequences of both human and viral origin is a consequence of HIV-1’s propensity to integrate into introns of transcriptionally active genes. These transcripts are an HIV-1 RNA footprint that may provide the means for expression of Tat in latently infected cells. Here, the HIV-1 RNA footprint in the CCL19-induced primary cell model of HIV-1 latency and in ex vivo samples from individuals on suppressive therapy was studied using target enrichment and next-generation sequencing technologies. Chimeric cellular:tat mRNAs were detected in the primary cell model that had some stretches of tat sequence incorporated in a variety of different configurations. A subset of these have the potential to translate Tat in their original unfragmented forms. In addition, as expected for the generation of readthrough transcripts, HIV-1 integration was predominantly parallel to the human gene, although a small proportion was attributed to the convergent orientation. No cellular:tat mRNAs were detected in the ex vivo samples, however, 3’ LTR activation and the use of the splice donor 1 (SD1) site were the major mechanisms leading to the generation of chimeras. The restrictive context of incorporation of tat sequences into a chimeric cellular:HIV transcript would impede translation through canonical 5’ cap-dependent ribosome scanning modalities. An internal ribosome entry site (IRES) located within Tat encoding sequences has been described and its properties in the context of chimeric cellular:tat mRNA was investigated. Robust, but low-level expression of Tat from an IRES-dependent mechanism was observed using luciferase-based assay systems, and a correspondingly weak reactivation of viral production from the J-Lat10.6 T-cell line model of HIV-1 latency was detected. In addition, SRP14 and HMGB3, two cellular RNA-binding proteins, are putative co-factors of Tat IRES translation detected by affinity purification-mass spectrometry, were shown to be positive and negative regulators of Tat expression respectively and may have roles in the regulation of HIV replication. In cells with quiescent proviruses this novel pathway of Tat expression could be targeted as part of a more biologically relevant combinatorial strategy for reversing latency.
    Keywords
    HIV-1; tat; translational regulation; latency; RNA processing; readthrough transcripts

    Export Reference in RIS Format     

    Endnote

    • Click on "Export Reference in RIS Format" and choose "open with... Endnote".

    Refworks

    • Click on "Export Reference in RIS Format". Login to Refworks, go to References => Import References


    Collections
    • Minerva Elements Records [45770]
    • Microbiology & Immunology - Theses [178]
    • Doherty Institute - Theses [30]
    Minerva AccessDepositing Your Work (for University of Melbourne Staff and Students)NewsFAQs

    BrowseCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects
    My AccountLoginRegister
    StatisticsMost Popular ItemsStatistics by CountryMost Popular Authors