?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.relation=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F8635%2F&rft.title=Novel+Mechanisms+of+Resistance+to+HIV-1+Reverse+Transcriptase+(RT)+Inhibitors%3A+A+Molecular+and+Clinical+Characterization+of+Mutations+in+the+Connection+and+RNase+H+Domains+of+RT&rft.creator=Brehm%2C+Jessica+Holly&rft.description=Current+antiretroviral+therapy+(ART)+has+reduced+morbidity+and+mortality+from+HIV-1+infection%2C+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%2C+attention+has+focused+on+the+connection+and+RNase+H+domains+of+RT+as+locations+of+drug+resistance+mutations%2C+but+the+prevalence%2C+molecular+mechanisms%2C+and+impact+of+such+mutations+on+response+to+ART+are+uncertain.+We+therefore+performed+a+series+of+studies+to+address+this+uncertainty%2C+including+in+vitro+selection+of+HIV-1+resistant+to+3'-azidothymidine+(AZT)%2C+drug+susceptibility+studies%2C+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%2C+we+provide+several+lines+of+evidence+that+connection+and+RNase+H+domain+mutations+emerge+with+ART+and+impact+nucleoside%2Fnucleotide+reverse+transcriptase+inhibitor+(NRTI)+susceptibility.+First%2C+the+connection+domain+mutation+A371V+and+the+RNase+H+domain+mutation+Q509L+are+selected+in+vitro+with+AZT+and+confer+%3E+50-fold+AZT+resistance+and+low-level+cross+resistance+to+lamivudine%2C+abacavir+and+tenofovir+when+in+the+context+of+thymidine+analog+mutations+(TAMs)+in+the+polymerase+domain+of+RT.+Second%2C+we+show+that+mutation+Q509L+in+the+RNase+H+domain+promotes+dissociation+of+RT+from+RNA%2FDNA+template%2Fprimer+bound+in+an+RNase+H+competent+mode%2C+thereby+decreasing+secondary+RNase+H+cleavage+and+destruction+of+the+template%2Fprimer.+As+a+consequence%2C+template%2Fprimer+binds+in+a+polymerase+competent+mode+allowing+AZT-monophosphate+excision%2C+DNA+polymerization+and+AZT+resistance.+Third%2C+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%2C+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%2C+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%2Flamivudine%2Fstavudine+or+nevirapine%2Flamivudine%2Fstavudine+when+virologic+failure+is+defined+as+confirmed+plasma+HIV-1+RNA+%3E+1%2C000+copies%2FmL.+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.&rft.date=2010-09-28&rft.type=University+of+Pittsburgh+ETD&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F8635%2F1%2FBrehmJ_Thesis_15Sep2010_FINAL.pdf&rft.identifier=++Brehm%2C+Jessica+Holly++(2010)+Novel+Mechanisms+of+Resistance+to+HIV-1+Reverse+Transcriptase+(RT)+Inhibitors%3A+A+Molecular+and+Clinical+Characterization+of+Mutations+in+the+Connection+and+RNase+H+Domains+of+RT.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++