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Dissertation
Interactions between HIV-1 and host cell RNAi pathways and the characterization of a conditional promoter for use in RNAi-based anti-HIV-1 therapeutics
Doctor of Philosophy (Ph.D.), Drexel University
Dec 2010
DOI:
https://doi.org/10.17918/00007652
Abstract
RNA interference is an evolutionarily conserved mechanism of gene regulation with a role in antiviral defense in invertebrates. It has been of interest to discover whether it serves a similar antiviral function in vertebrates. Considerable evidence indicates that RNAi serves an antiviral role during HIV-1 infections and, in turn, HIV-1 has evolved strategies to suppress the silencing machinery. HIV-1 was initially proposed to accomplish this by utilizing the viral products, Tat and TAR, to inhibit the miRNA processing activity of Dicer and to sequester the Dicer co-factor, TRBP, respectively. However, these findings have been controversial and we have re-examined this potentially important interaction. Our results support and extend the conclusion that Tat does not reduce the silencing capacity of cells. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR element do not reduce the efficacy of silencing. TRBP is of particular interest with regard to HIV-1 replication and RNAi. In addition to its role as a co-factor for Dicer in miRNA processing, it binds with high affinity to the HIV-1 TAR element and can increase the efficiency of expression of HIV-1 gene products. While multiple mechanisms explaining the role of TRBP in HIV-1 replication have been proposed, our data show that TRBP promotes HIV-1 primarily by averting the PKR-mediated antiviral immunity. Moreover, the effect of TRBP on HIV-1 replication is independent of its role in the RNAi pathway. Additionally, we have characterized a chimeric promoter, consisting of a chicken [beta]-actin core promoter fused to the viral TAR element, for the conditional expression of interfering RNAs against HIV-1 for use in the development of antiviral therapeutics. This hybrid CK-TAR promoter can induce potent levels of silencing in response to Tat produced from co-transfected plasmids or during viral replication. Furthermore, compared to the Tat-dependent HIV-1 LTR, it is less responsive to extracellular stimuli including LPS, TNF[alpha], and PMA. The Tat-inducible CK-TAR promoter is an alternative to the LTR that can minimize the risk of vector mobilization and can drive polycistronic expression of interfering RNAs.
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Details
- Title
- Interactions between HIV-1 and host cell RNAi pathways and the characterization of a conditional promoter for use in RNAi-based anti-HIV-1 therapeutics
- Creators
- Viraj R. Sanghvi
- Contributors
- Laura F. Steel (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xix, 224 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991021889001504721