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Mechanisms by which Nonnucleoside Reverse Transcriptase Inhibitors Block HIV-1 Replication Alone and in Combination with other Reverse Transcriptase Inhibitors

Radzio, Jessica Ann (2010) Mechanisms by which Nonnucleoside Reverse Transcriptase Inhibitors Block HIV-1 Replication Alone and in Combination with other Reverse Transcriptase Inhibitors. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

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 5'end 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.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Radzio, Jessica Annjradzio@hotmail.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSluis-Cremer, Nicolasnps2@pitt.eduNPS2
Committee MemberMellors, Johnjwm1@pitt.eduJWM1
Committee MemberWang, Tianyitywang@pitt.eduTYWANG
Committee MemberAyyavoo, Velpandivelpandi@pitt.eduVELPANDI
Committee MemberAmbrose, Zandreazaa4@pitt.eduZAA4
Date: 29 September 2010
Date Type: Completion
Defense Date: 13 August 2010
Approval Date: 29 September 2010
Submission Date: 26 July 2010
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: HIV-1; nonnucleoside reverse transcriptse inhibitors; reverse transcriptase
Other ID: http://etd.library.pitt.edu/ETD/available/etd-07262010-165005/, etd-07262010-165005
Date Deposited: 10 Nov 2011 19:54
Last Modified: 15 Nov 2016 13:47
URI: http://d-scholarship.pitt.edu/id/eprint/8628

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