eprintid: 8772 rev_number: 5 userid: 6 dir: disk0/00/00/87/72 datestamp: 2011-11-10 19:55:37 lastmod: 2016-11-15 13:47:39 status_changed: 2011-11-10 19:55:37 type: thesis_degree metadata_visibility: show contact_email: epiphany-00@hotmail.com item_issues_count: 0 eprint_status: archive creators_name: Clark, Shauna Antoinette creators_email: sac2@pitt.edu; epiphany-00@hotmail.com creators_id: SAC2 title: Distinct Mechanisms of HIV-1 Hypersusceptibility to Non-Nucleoside Reverse Transcriptase Inhibitors: A Discussion of Nucleoside Reverse Transcriptase Inhibitor Induced Mutations V118I, H208Y, and T215Y ispublished: unpub divisions: sch_gsph_infectiousdiseasesmicrobiology full_text_status: public keywords: Drug resistance; HIV-1; Hypersusceptibility; NNRTI abstract: 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. date: 2008-09-28 date_type: completed institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Mellors, John etdcommittee_name: Cascio, Michael etdcommittee_name: Sluis-Cremer, Nicolas etdcommittee_name: Gupta, Phalguni etdcommittee_email: mellors@dom.pitt.edu etdcommittee_email: cascio@pitt.edu etdcommittee_email: nps2@pitt.edu etdcommittee_email: pgupta@pitt.edu etdcommittee_id: JWM1 etdcommittee_id: CASCIO etdcommittee_id: NPS2 etdcommittee_id: PGUPTA etd_defense_date: 2008-07-17 etd_approval_date: 2008-09-28 etd_submission_date: 2008-07-31 etd_access_restriction: 5_year etd_patent_pending: FALSE assigned_doi: doi:10.5195/pitt.etd.2011.8772 thesis_type: dissertation degree: PhD committee: John Mellors, MD (mellors@dom.pitt.edu) - Committee Chair committee: Michael Cascio, PhD (cascio@pitt.edu) - Committee Member committee: Nicolas Sluis-Cremer, PhD (nps2@pitt.edu) - Committee Member committee: Phalguni Gupta, PhD (pgupta@pitt.edu) - Committee Member etdurn: etd-07312008-150934 other_id: http://etd.library.pitt.edu/ETD/available/etd-07312008-150934/ other_id: etd-07312008-150934 citation: Clark, Shauna Antoinette (2008) Distinct Mechanisms of HIV-1 Hypersusceptibility to Non-Nucleoside Reverse Transcriptase Inhibitors: A Discussion of Nucleoside Reverse Transcriptase Inhibitor Induced Mutations V118I, H208Y, and T215Y. Doctoral Dissertation, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/8772/1/ClarkSdissertation9.08.pdf