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Activity Profiles & Mechanisms of Resistance of 3’-Azido-2’,3’-Dideoxynucleoside Analog Reverse Transcriptase Inhibitors of HIV-1

Meteer, Jeffrey (2013) Activity Profiles & Mechanisms of Resistance of 3’-Azido-2’,3’-Dideoxynucleoside Analog Reverse Transcriptase Inhibitors of HIV-1. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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To investigate mechanisms of HIV-1 resistance to 3’-azidonucleoside analog reverse transcriptase inhibitors, in vitro selection experiments were conducted by serial passage of HIV-1LAI in MT-2 cells in increasing concentrations of 3′-azido-2′,3′-dideoxyguanosine (3′-azido-ddG), 3′-azido-2′,3′-dideoxycytidine (3′-azido-ddC), or 3′-azido-2′,3′-dideoxyadenosine (3′-azido-ddA). 3′-Azido-ddG selected for virus 5.3-fold resistant to 3′-azido-ddG. Population sequencing of the reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. Site-directed mutagenesis showed that these 5 mutations only conferred ~2.0-fold resistance. Single-genome sequencing analyses revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2- to 4.0-fold 3′-azido-ddG resistance. By contrast, 3′-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance. We were unable to select HIV-1 resistant to 3′-azido-ddA, even at concentrations of 3′-azido-ddA that yielded high intracellular 3′-azido-ddA-5′-triphosphate levels. We have also defined the molecular mechanisms of 3’-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V/F77L/V106I/L214F/R277K/K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3’-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows HIV-1 RT to effectively discriminate between the natural nucleotide (dGTP) and 3’-azido-ddG-triphosphate (3’-azido-ddGTP). 3’-azido-ddGTP discrimination was primarily driven by a decrease in 3’-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT’s ability to excise the chain-terminating 3’-azido-ddG-monophosphate (3’-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme’s ability to excise 3’-azido-ddGMP on an RNA/DNA, but not on DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Taken together, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3’-azido-2’,3’-dideoxynucleosides that can be exploited in the design of novel nucleoside RT inhibitors.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairMellors, Johnjwm1@pitt.eduJWM1
Committee MemberSluis-Cremer, Nicolasnps2@pitt.eduNPS2
Committee MemberKhan, Saleemkhan@pitt.eduKHAN
Committee MemberKinchington, Paulkinchingtonp@upmc.eduKINCH
Committee MemberJohnson, Danieljohnsond@pitt.eduJOHNSOND
Date: 29 August 2013
Date Type: Publication
Defense Date: 18 July 2013
Approval Date: 29 August 2013
Submission Date: 19 August 2013
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 151
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Virology and Microbiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: HIV Reverse Transcriptase NRTI 3'-azido Antiretroviral Nucleoside Analog
Date Deposited: 29 Aug 2013 18:52
Last Modified: 19 Dec 2016 14:41


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