In 2012 35.3 million people worldwide were infected with human Immunodeficiency Virus Type-1 (HIV-1). Although no vaccine exists, highly active anti-retroviral therapy (HAART) has enabled patients to maintain HIV plasma concentrations at undetectable levels. Regrettably, HARRT has been unsuccessful in eliminating latent reservoirs resulting in a persistent infection that could potentially generate escape mutations. We have been investigating the suitability of trans-splicing group I introns (GIIs) as anti-viral agents. GIIs are catalytic RNA molecules that can splice an RNA sequence of choice (3′-exon) to a target such as HIV derived RNAs. Splice-product is a hybrid molecule of 5′-target sequence and 3′-exon sequence. Our anti-HIV-1 GIIs target two highly conserved regions of the HIV-1 genome, the primer activation signal (PAS) and the primer binding site (PBS). All viral associated RNA species generated within an HIV-1 infected cell contain the PAS/PBS sequences. We have designed five anti-HIV GIIs designated: PAS126, PAS126L, PAS128, PAS128L and PBS182. Four GIIs exhibited splicing as revealed by RT-PCR and sequencing analysis. By fusing the Gaussia luciferase coding sequence to target RNAs we demonstrate that GIIs can mediate a ~70% reduction in observed luminescence. We also demonstrate that a 3′-exon encoding the pro-apoptotic protein, ΔN-Bax, can induce apoptosis and augments viral suppression. Transient transfection of each GII into HEK 293T cells, followed by infection with pseudoVSV-G-HIVNL4-3 revealed ~50% viral suppression as measured by p24 ELISA. Transformed HEK 293T cells, expressing PAS128L (with and without ΔN-Bax), were infected and assayed for the presence of splice-product. Canonical splice-product and a cryptic splice-variant were detected. Sequence analysis of the cryptic splice-variant revealed that targeting remained highly specific to the PAS/PBS sequences. These transformed cell lines, a heterogeneous population, exhibited ~2 logs viral suppression. Clonal cell lines were infected and assayed for caspase-3 activity, and confirmed that expression of ΔN-Bax induced apoptosis upon challenge with HIV. No HIV was detected by p24 ELISA analysis, demonstrating that a homogenous population of GII expressing cells are capable of 100% viral suppression.
|Author||Jamie L. Dawson|
|Contributor||Mary Ann McDowell, Committee Member|
|Contributor||Malcolm Fraser, Committee Chair|
|Contributor||Paul Huber, Committee Member|
|Contributor||Joseph OTousa, Committee Member|
|Degree Level||Doctoral Dissertation|
|Degree Discipline||Biological Sciences|
|Degree Name||Doctor of Philosophy|
|Departments and Units|
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