Dissertation
The positive regulation of HIV-1 Vif mRNA splicing is required for efficient virus replication
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2009
DOI: 10.17077/etd.0rsvk6qc
Abstract
<p>Productive HIV-1 transcription yields a single ∼9.2kb RNA. From this ∼9.2kb genomic RNA, greater than 40 different subgenomic mRNAs can be produced through alternative splicing using four 5' splice sites (ss) and seven 3'ss. Splice site utilization is governed by the inherent strength of the splice sites and by several identified <em>cis</em> acting elements. The HIV-1 Vif protein, required to overcome the cellular antiviral factor APOBEC3G, is encoded by a singly-spliced mRNA coupling 5'ss D1 to 3'ss A1. Alternatively, mRNAs spliced at A1 can utilize a downstream 5'ss, D2, resulting in inclusion of non-coding exon 2 in a small percentage of mRNAs. Expression of <em>vif</em> mRNA within infected cells is required but maintained at low levels. The purpose of studies described in this thesis was to identify and characterize elements within the HIV-1 genome regulating <em>vif</em> mRNA splicing. We identified an exonic splicing enhancer (ESE) within the 18nt downstream of HIV-1 3'ss A1, ESEVif. Mutation of ESEVif within the HIV-1 proviral clone pNL4-3 resulted in a dramatic decrease in <em>vif</em> mRNA, Vif protein, and undetectable levels of non-coding exon 2 inclusion. The cellular splicing factor SRp75 was found to selectively bind ESEVif <em>in vitro</em>. ESEVif mutant virus replicated in APOBEC3G-deficient T-cell lines as efficiently as wild-type virus. In APOBEC3G-producing T-cell lines, ESEVif mutant virus replicated to lower levels than wild-type virus. Other studies have identified additional mRNA splicing elements regulating splicing at A1: the downstream 5'ss D2 can promote or repress splicing, a G4 motif downstream of D2 represses splicing, and two ASF/SF2 dependent splicing enhancers, ESEM1 and ESEM2, promote splicing. Mutational analysis described in this thesis determined that loss of both ESEVif and the G4 motif resulted in wild-type levels of splicing at A1. Mutations of each identified ESE influenced <em>vif</em> mRNA splicing in the order ESEVif>ESEM2>ESEM1. The data presented in this thesis support a model of <em>vif</em> mRNA splicing regulation in which exon 2 ESE act to overcome the negative G4 motif insuring sufficient levels of Vif production for efficient replication in the presence of APOBEC3G.</p>
Details
- Title: Subtitle
- The positive regulation of HIV-1 Vif mRNA splicing is required for efficient virus replication
- Creators
- Colin Michael Exline - University of Iowa
- Contributors
- C. Martin Stoltzfus (Advisor)Stanley Perlman (Committee Member)Richard Roller (Committee Member)Andrew Russo (Committee Member)Steven Varga (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Autumn 2009
- DOI
- 10.17077/etd.0rsvk6qc
- Publisher
- University of Iowa
- Number of pages
- xv, 143 pages
- Copyright
- Copyright 2009 Colin Michael Exline
- Language
- English
- Description bibliographic
- Includes bibliographical references (pages 121-143).
- Academic Unit
- Microbiology and Immunology; Craniofacial Anomalies Research Center
- Record Identifier
- 9983776894802771
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