Inhibition of reverse transcriptase (RT) is a vital tactic in the prevention of human immunodeficiency virus 1 (HIV-1). Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a class of compounds demonstrated to act as allosteric inhibitors of RT DNA polymerization. However, several lines of evidence suggest that polymerization may not be the main mechanism of inhibition of reverse transcription. It has been demonstrated that NNRTIs also have the ability to modulate RT ribonuclease (RNase) H cleavage. Additionally, recent evidence suggests that resistance to chain-terminating nucleoside reverse transcriptase inhibitors (NRTIs) is dependent on a balance between the polymerase and RNase H activities of the enzyme. In light of this, I hypothesize that NNRTIs block reverse transcription by exerting effects on both the DNA polymerase and RNase H active sites of the enzyme, significantly disrupting the equilibrium between these two enzymatic activities. Therefore, the ability for NNRTIs to be combined with other classes of RT inhibitors in antiretroviral therapies will depend on how these compounds respond to the NNRTI-induced shift in the polymerase/RNase H activity equilibrium. This study demonstrates that NNRTIs cause the accelerated appearance of secondary RNase H cleavage products that have decreased RNA/DNA hybrid structures. As a result, these template/primers(T/Ps) are not sufficient substrates for NRTI removal and therefore, excision is less efficient in the presence of NNRTIs. Additionally, fluorescent resonance energy transfer experiments demonstrate that NNRTIs cause a shift in the binding of RT and T/P such that the RNase H domain is moved away from the 5end of the primer. Finally, subunit-specific analysis shows that resistance to RTI combination therapy facilitated by the N348I mutation is a result of effects from the p51 subunit. I propose that the binding of NNRTIs cause RT to bind to T/P in a polymerase-incompetent mode, resulting in decreased polymerization and shorted RNase H cleavage products. Additionally, N348I can facilitate dual resistance by favoring the polymerase-competent binding mode. This work is of public health significance because it lays the foundation for the development of new reverse transcriptase inhibitors and highlights the importance of resistance in the connection domain of HIV-1 RT.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07262010-165005 |
| Date | 29 September 2010 |
| Creators | Radzio, Jessica Ann |
| Contributors | Tianyi Wang, PhD, Velpandi Ayyavoo, PhD, John Mellors, MD, Nicolas Sluis-Cremer, PhD, Zandrea Ambrose, PhD |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07262010-165005/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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