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(Unpublished)
Abstract
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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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| 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 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/19693 |
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