Hypersusceptibility (HS) to non-nucleoside reverse transcriptase inhibitors (NNRTI) improves virological response to NNRTI-containing regimens. NNRTI HS is associated with nucleoside RT inhibitor (NRTI) mutations, especially those conferring resistance to AZT. Recent logistic regression analyses of a large genotype-phenotype dataset showed the NRTI mutations most strongly associated with NNRTI HS are T215Y, H208Y and V118I. We hypothesized that NRTI mutations V118I, H208Y, and T215Y in combination cause NNRTI HS and that this phenotype is due to multiple mechanisms including a decrease in enzyme activity and/or deficient viral replication due to decreased virion packaged RT. Therefore we sought to: (1) determine the phenotypic effects of these mutations alone and in combination on the susceptibilityof infectious molecular clones and recombinant reverse transcriptase proteins to efavirenz, delavirdine, and nevirapine; and (2) elucidate differences in viral replication, protein production and packaging for virus containing mutations V118I, H208Y, and/or T215Y. We established different patterns of NNRTI HS and replication capacity depending on the combination of mutations present. HIV-1 virus containing H208Y + T215Y were HS to all NNRTI; whereas the V118I/T215Y virus was only HS to delavirdine and nevirapine. H208Y + T215Y viruses exhibited reduced replication capacity compared to wildtype HIV-1. In comparison, the V118I/T215Y virus replicated as efficiently as wildtype virus. Upon further investigation we found the amount of HIV-1 RT incorporated into the H208Y + T215Y viruses was significantly reduced compared with wildtype virus due to decreased viral packaging of GagPol precursors. Interestingly, the RT content in the V118I/T215Y virus was similar to wildtype virus. Furthermore, purified recombinant RT containing the H208Y+ T215Y mutations were not NNRTI HS. In contrast, the V118I/T215Y mutant RT showed five-fold increased susceptibility to NNRTI. Our work highlights the complexity of the HS phenotype and provides an in-depth understanding of how NRTI mutations V118I, H208Y, and T215Y contribute to increased NNRTI susceptibility.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07312008-150934 |
| Date | 28 September 2008 |
| Creators | Clark, Shauna Antoinette |
| Contributors | Phalguni Gupta, PhD, Nicolas Sluis-Cremer, PhD, Michael Cascio, PhD, John Mellors, MD |
| 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-07312008-150934/ |
| 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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