eprintid: 8635 rev_number: 6 userid: 6 dir: disk0/00/00/86/35 datestamp: 2011-11-10 19:54:12 lastmod: 2016-11-15 13:47:03 status_changed: 2011-11-10 19:54:12 type: thesis_degree metadata_visibility: show contact_email: wilddaisyo@yahoo.com item_issues_count: 0 eprint_status: archive creators_name: Brehm, Jessica Holly creators_email: wilddaisyo@yahoo.com title: Novel Mechanisms of Resistance to HIV-1 Reverse Transcriptase (RT) Inhibitors: A Molecular and Clinical Characterization of Mutations in the Connection and RNase H Domains of RT ispublished: unpub divisions: sch_gsph_infectiousdiseasesmicrobiology full_text_status: public keywords: A371V; antiretroviral therapy (ART); AZT-MP excision; HIV-1 subtype B; HIV-1 subtype C; N348I; NNRTI; NRTI; Q509L; RNase H cleavage; RT RNase H domain; A360V; drug resistance; nucleoside/tide reverse transcriptase inhibitor; RT connection domain; Human Immunodeficiency Virus - type 1(HIV-1); non-nucleoside reverse transcriptase inhibitor; reverse transcriptase (RT) abstract: 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. date: 2010-09-28 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_type: committee_member etdcommittee_name: Mellors, John W etdcommittee_name: Martinson, Jeremy etdcommittee_name: Yeh, Joanne etdcommittee_name: Sluis-Cremer, Nicolas etdcommittee_name: Gupta, Phalguni etdcommittee_email: jwm1@pitt.edu etdcommittee_email: jmartins@pitt.edu etdcommittee_email: jiyeh@pitt.edu etdcommittee_email: nps2@pitt.edu etdcommittee_email: pgupta1@pitt.edu etdcommittee_id: JWM1 etdcommittee_id: JMARTINS etdcommittee_id: JIYEH etdcommittee_id: NPS2 etdcommittee_id: PGUPTA1 etd_defense_date: 2010-06-29 etd_approval_date: 2010-09-28 etd_submission_date: 2010-07-26 etd_access_restriction: 5_year etd_patent_pending: FALSE assigned_doi: doi:10.5195/pitt.etd.2011.8635 thesis_type: dissertation degree: PhD committee: John W. Mellors (jwm1@pitt.edu) - Committee Chair committee: Jeremy Martinson (jmartins@pitt.edu)- Committee Member committee: Joanne Yeh (jiyeh@pitt.edu) - Committee Member committee: Nicolas Sluis-Cremer (nps2@pitt.edu) - Committee Member committee: Phalguni Gupta (pgupta1@pitt.edu) - Committee Member etdurn: etd-07262010-212223 other_id: http://etd.library.pitt.edu/ETD/available/etd-07262010-212223/ other_id: etd-07262010-212223 citation: Brehm, Jessica Holly (2010) Novel Mechanisms of Resistance to HIV-1 Reverse Transcriptase (RT) Inhibitors: A Molecular and Clinical Characterization of Mutations in the Connection and RNase H Domains of RT. Doctoral Dissertation, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/8635/1/BrehmJ_Thesis_15Sep2010_FINAL.pdf