Regulation of HIV-1 Splicing Public Deposited

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  • March 21, 2019
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  • Emery, Ann
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • ABSTRACT Ann Emery: Regulation of HIV-1 Splicing (Under the direction of Ronald Swanstrom) The Human Immunodeficiency Virus Type 1 (HIV-1) has a single primary transcript – full-length genomic RNA. Left unspliced, it serves as either genomic RNA or as mRNA for the viral reverse transcriptase, protease, integrase, and structural proteins. The mRNAs for all other viral proteins require splicing of the full-length transcript. HIV-1 undergoes a complex program of splicing and suppression of splicing to make more than 50 transcript types. Since these complex splicing patterns are essential for viral replication, splicing disruption could be a point of vulnerability given a detailed understanding of the steps involved. In order to assess the regulation of splicing, the products of splicing have to be quantifiable. This dissertation describes two Primer ID-tagged deep sequencing assays developed to quantify HIV-1 splicing in the context of viral infection/transfection and in the context of a full-length viral genome. The depth of sequencing allows quantification of even rare splicing events. Using these deep sequencing assays I examined splicing across HIV-1 subtypes and between HIV-1 and SIVmac239 and found that while patterns of splicing are well conserved, wide variation in acceptor usage is tolerated among different HIV-1 strains. Correct splicing depends on cis-acting control sequences interacting with cellular splicing factors. I quantified the effects of mutations that alter these sequences or the secondary RNA structures containing them. I reevaluated the effects of previously characterized splicing regulatory sequences, validating the functions of some but redefining others. In collaborative efforts I showed that mutation of splicing factor binding sites or knock down of cellular splicing proteins produces specific splicing phenotypes. Analysis of a set of global silent mutations across the HIV-1 genome found regions of the viral genome that interfere with suppression of splicing and carry a high fitness cost. These experiments show that HIV-1 splicing regulation involves more complex patterns of factor binding and cooperative interactions than previously described, suggesting that existing models are overly simplistic, and my studies contribute to a more accurate description and understanding of HIV-1 splicing.
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  • In Copyright
Advisor
  • Prins, Jan
  • Jones, Corbin
  • Moorman, Nathaniel
  • Weeks, Kevin
  • Swanstrom, Ronald
  • Mieczkowski, Piotr
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017
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