Novel Anti-Viral Strategies for Lipid-Enveloped Viruses

Doctoral Dissertation
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Abstract

Apoptotic mimicry utilized by lipid-enveloped viruses for infection is dependent on external leaflet viral phosphatidylserine (PS) expression and host cell kinase activity involving TIM proteins, AXL, and TYRO3. In addition, it has been shown that matrix proteins Ebola VP40 and HIV-1-Gag induce external leaflet expression of PS. UCN-01 is a staurosporine FDA approved for phase II clinical trials for multiple neoplasms, which acts as a non-specific kinase inhibitor and has been shown to disrupt PS localization to the plasma membrane by redistributing to endomembranes. By utilizing UCN-01 at lower concentrations than used for anti-cancer clinical trials it is hypothesized that viral replication in multiple lipid enveloped viruses can be inhibited in two ways: first, by inhibiting matrix protein localization to the plasma membrane and viral budding by localizing PS to endomembranes from the plasma membrane, and second, by inhibiting viral infection which utilizes apoptotic mimicry by reducing available PS on viral particles and by also inhibiting host cell kinase activity of receptor proteins known to interact with PS. Cellular toxicity of UCN-01 was reproduced from the literature and concentrations from 50-400nM were determined to be within the acceptable range in the cells being studied. Using these concentrations UCN-01 was shown to reduce plasma membrane localization of Ebola VP40, Marburg VP40, and HIV-1-Gag significantly using confocal imaging of recombinant GFP tagged viral proteins. Changes in budding were then assessed in Marburg VP40 and Ebola VP40 using western blot with significant increases in budding at 200 nM and 300 nM in Ebola VP40 at 24 hours and significant decreases in budding at 100 nM – 400 nM in Ebola VP40 and 400 nM in Marburg VP40 at 48 hours. TEM of Ebola VP40 showed reduction of surface budding in presence of UCN-01 and 7-OSS and a non-significant decrease in VLP diameter with 100 nM UCN-01 treatment and a significant decrease in VLP diameter with 50 nM 7-OSS treatment. A decrease of filament density on the cell surface was also shown using SEM for 100 nM UCN-01 and 50 nM 7-OSS treatments. Finally, live Chikungunya virus, Zika virus, HIV-1, and Ebola virus was shown to be inhibited by UCN-01 treatment. Given the success seen in these models, additional live virus studies will be pursued, including Dengue virus and Marburg virus among other lipid enveloped viruses. Current anti-HIV methodologies have allowed for the reduction of HIV plasma concentrations to undetectable levels in infected patients, but have been unable to eliminate latent reservoirs, resulting in a persistent infection that could potentially generate escape mutations. We have created trans-splicing group I introns targeting the Primer Activation Signal and the Primer Binding Site of HIV as potential transgene antiviral agents in an effort to prevent re-establishment of virus pathology in the absence of drug therapy. Trans-splicing group I introns are catalytic RNA molecules that can splice an RNA sequence of choice (3’-exon) to a target RNA molecule, in this case creating a hybrid RNA molecule that encodes a protein which is capable of inhibiting virus replication. We demonstrate the catalytic capabilities of these group I introns against RNA derived from infecting virus (in addition to an artificial target) and that inclusion of a 3’-exon encoding the pro-apoptotic protein ∆N-Bax induces programmed cell death, augmenting the suppression of viral replication. All introns exhibited specific targeting and splicing of RNAs containing PAS/PBS sequences as demonstrated by RT-PCR and sequencing analysis. By fusing the Gaussia luciferase coding sequence to target RNAs containing the PAS/PBS sequence we were able to demonstrate that these introns mediated a ~70% reduction in observed luminescence produced from transfected cells. Annexin V staining demonstrated efficient induction of apoptotic cell death resulting from expression of ∆N-Bax from splice products. Transient transfection of each intron in HEK-293T cells, followed by infection with pseudoVSV-G-HIVNL4-3 at MOI 2 suppressed viral replication by ~50% at 4 dpi as measured by p24 ELISA. Clonal CEM-A cell lines were generated by limited dilution and confirmed for group I intron via RT-PCR and ∆N-Bax expression by Caspase 3 assay. Clones were then infected at MOI 1 with HIV-1 pNL4-3 and at 14 dpi p24 ELISA analysis yielded no detectable HIV in a clone and 2 logs reduction in multiple clones, demonstrating that a homogeneous population of group I intron expressing cells are capable of 100% viral suppression. In addition, two clones showed 2 logs reduction at 30 dpi. Future experiments include moving into an animal model to see if viral clearance is achievable in vivo.

Attributes

Attribute NameValues
Author Mark Evan Fraser
Contributor Robert Stahelin, Research Director
Contributor Shaun Lee, Research Director
Degree Level Doctoral Dissertation
Degree Discipline Integrated Biomedical Sciences
Degree Name Doctor of Philosophy
Banner Code
  • PHD-IBBI

Defense Date
  • 2019-03-28

Submission Date 2019-04-05
Record Visibility and Access Public
Content License
  • All rights reserved

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