Trans-Splicing Group I Introns Targeting Highly Conserved HIV-1 Sequences Coupled with Induction of Apoptosis Are Effective Mediators of Viral Suppression

Doctoral Dissertation

Abstract

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.

Attributes

Attribute NameValues
URN
  • etd-04172015-131931

Author Jamie L. Dawson
Advisor Malcolm Fraser
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 PhD
Defense Date
  • 2015-04-01

Submission Date 2015-04-17
Country
  • United States of America

Subject
  • Apoptosis

  • HIV

  • Group I Intron

  • Bax

  • Virus

Publisher
  • University of Notre Dame

Language
  • English

Record Visibility and Access Public
Content License
  • All rights reserved

Departments and Units

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