Current antiretroviral therapy (ART) has reduced morbidity and mortality from HIV-1 infection, but the long-term efficacy of ART is limited by selection of HIV-1 drug-resistant variants. Most HIV-1 drug resistance mutations that have been studied are located in the polymerase domain of HIV-1 reverse transcriptase (RT) and this region of RT is sequenced in genotyping tests used clinically to guide ART. Recently, attention has focused on the connection and RNase H domains of RT as locations of drug resistance mutations, but the prevalence, molecular mechanisms, and impact of such mutations on response to ART are uncertain. We therefore performed a series of studies to address this uncertainty, including in vitro selection of HIV-1 resistant to 3-azidothymidine (AZT), drug susceptibility studies, biochemical assays and genotype analysis of clinical samples to identify and characterize resistance mutations in the RT connection and RNase H domains. From this work, we provide several lines of evidence that connection and RNase H domain mutations emerge with ART and impact nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) susceptibility. First, the connection domain mutation A371V and the RNase H domain mutation Q509L are selected in vitro with AZT and confer > 50-fold AZT resistance and low-level cross resistance to lamivudine, abacavir and tenofovir when in the context of thymidine analog mutations (TAMs) in the polymerase domain of RT. Second, we show that mutation Q509L in the RNase H domain promotes dissociation of RT from RNA/DNA template/primer bound in an RNase H competent mode, thereby decreasing secondary RNase H cleavage and destruction of the template/primer. As a consequence, template/primer binds in a polymerase competent mode allowing AZT-monophosphate excision, DNA polymerization and AZT resistance. Third, the connection domain mutation A360V emerges in patients after prolonged exposure to AZT monotherapy and increases resistance to AZT in the context of 3 or more TAMs. Fourth, connection and RNase H domain mutations are not more frequent at virologic failure in HIV-1 subtype B infected patients treated with 2 NRTI plus efavirenz when failure is defined as a small increase in plasma HIV-1 RNA. However, the connection domain mutation N348I emerges frequently at virologic failure in HIV-1 subtype C infected patients in South Africa who were treated with efavirenz/lamivudine/stavudine or nevirapine/lamivudine/stavudine when virologic failure is defined as confirmed plasma HIV-1 RNA > 1,000 copies/mL. This work provides strong evidence that RT connection and RNase H domain mutations emerge in HIV-1 infected patients treated with ART and these mutations are missed with currently available genotype tests. Mutations missed by routine genotyping tests pose a potential public health threat if left undetected and transmitted to others.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07262010-212223 |
| Date | 28 September 2010 |
| Creators | Brehm, Jessica Holly |
| Contributors | John W. Mellors, Phalguni Gupta, Joanne Yeh, Jeremy Martinson, Nicolas Sluis-Cremer |
| 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-07262010-212223/ |
| Rights | restricted, 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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