Title
The positive regulation of HIV-1 Vif mRNA splicing is required for efficient virus replication
Date of Degree
2009
Document Type
dissertation
Degree Name
PhD (Doctor of Philosophy)
Department
Microbiology
First Advisor
C. Martin Stoltzfus
Abstract
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 cis 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 vif 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 vif 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 vif mRNA, Vif protein, and undetectable levels of non-coding exon 2 inclusion. The cellular splicing factor SRp75 was found to selectively bind ESEVif in vitro. 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 vif mRNA splicing in the order ESEVif>ESEM2>ESEM1. The data presented in this thesis support a model of vif 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.
Pages
xv, 143
Bibliography
121-143
Copyright
Copyright 2009 Colin Michael Exline
Recommended Citation
Exline, Colin Michael. "The positive regulation of HIV-1 Vif mRNA splicing is required for efficient virus replication." dissertation, University of Iowa, 2009.
http://ir.uiowa.edu/etd/356.