Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1 Reverse Transcriptase Using a Transient Kinetic Analysis†
Identifieur interne : 002771 ( Istex/Corpus ); précédent : 002770; suivant : 002772Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1 Reverse Transcriptase Using a Transient Kinetic Analysis†
Auteurs : Adrian S. Ray ; Eisuke Murakami ; Aravind Basavapathruni ; Joseph A. Vaccaro ; Dagny Ulrich ; Chung K. Chu ; Raymond F. Schinazi ; Karen S. AndersonSource :
- Biochemistry [ 0006-2960 ] ; 2003.
Abstract
Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase (RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision, suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain terminator from an RNA−RNA primer−template may show how slight changes in selectivity against AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance. Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations. Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.
Url:
DOI: 10.1021/bi034435l
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Ray</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Murakami, Eisuke" sort="Murakami, Eisuke" uniqKey="Murakami E" first="Eisuke" last="Murakami">Eisuke Murakami</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Basavapathruni, Aravind" sort="Basavapathruni, Aravind" uniqKey="Basavapathruni A" first="Aravind" last="Basavapathruni">Aravind Basavapathruni</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Vaccaro, Joseph A" sort="Vaccaro, Joseph A" uniqKey="Vaccaro J" first="Joseph A." last="Vaccaro">Joseph A. 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Chu</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> The University of Georgia.</mods:affiliation></affiliation></author><author><name sortKey="Schinazi, Raymond F" sort="Schinazi, Raymond F" uniqKey="Schinazi R" first="Raymond F." last="Schinazi">Raymond F. Schinazi</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Emory University School of Medicine/Veterans Affairs MedicalCenter.</mods:affiliation></affiliation></author><author><name sortKey="Anderson, Karen S" sort="Anderson, Karen S" uniqKey="Anderson K" first="Karen S." last="Anderson">Karen S. Anderson</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. E-mail: karen.anderson@yale.edu. Phone: (203) 785-4526. Fax: (203) 785-7670.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:27E0B499B0C8B79DCFA4C54E465DD77F7037A1FA</idno><date when="2003" year="2003">2003</date><idno type="doi">10.1021/bi034435l</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-1F3PKRB5-X/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002771</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002771</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1
Reverse Transcriptase Using a Transient Kinetic Analysis<ref type="bib" target="#bi034435lAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Ray, Adrian S" sort="Ray, Adrian S" uniqKey="Ray A" first="Adrian S." last="Ray">Adrian S. Ray</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Murakami, Eisuke" sort="Murakami, Eisuke" uniqKey="Murakami E" first="Eisuke" last="Murakami">Eisuke Murakami</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Basavapathruni, Aravind" sort="Basavapathruni, Aravind" uniqKey="Basavapathruni A" first="Aravind" last="Basavapathruni">Aravind Basavapathruni</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Vaccaro, Joseph A" sort="Vaccaro, Joseph A" uniqKey="Vaccaro J" first="Joseph A." last="Vaccaro">Joseph A. Vaccaro</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: Department of Biochemistry, Tulane UniversityHealth Sciences Center, 1430 Tulane Ave. SL-43, New Orleans, LA70112.</mods:affiliation></affiliation></author><author><name sortKey="Ulrich, Dagny" sort="Ulrich, Dagny" uniqKey="Ulrich D" first="Dagny" last="Ulrich">Dagny Ulrich</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Chu, Chung K" sort="Chu, Chung K" uniqKey="Chu C" first="Chung K." last="Chu">Chung K. Chu</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> The University of Georgia.</mods:affiliation></affiliation></author><author><name sortKey="Schinazi, Raymond F" sort="Schinazi, Raymond F" uniqKey="Schinazi R" first="Raymond F." last="Schinazi">Raymond F. Schinazi</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Emory University School of Medicine/Veterans Affairs MedicalCenter.</mods:affiliation></affiliation></author><author><name sortKey="Anderson, Karen S" sort="Anderson, Karen S" uniqKey="Anderson K" first="Karen S." last="Anderson">Karen S. Anderson</name><affiliation><mods:affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</mods:affiliation></affiliation><affiliation><mods:affiliation> Yale University School of Medicine.</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. E-mail: karen.anderson@yale.edu. Phone: (203) 785-4526. Fax: (203) 785-7670.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2003</date><date type="published">2003</date><biblScope unit="vol">42</biblScope><biblScope unit="issue">29</biblScope><biblScope unit="page" from="8831">8831</biblScope><biblScope unit="page" to="8841">8841</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase (RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision, suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain terminator from an RNA−RNA primer−template may show how slight changes in selectivity against AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance. Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations. Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>rtaztr</json:string><json:string>aztmp</json:string><json:string>rtwt</json:string><json:string>next correct nucleotide</json:string><json:string>d4tmp</json:string><json:string>biochemistry</json:string><json:string>primer</json:string><json:string>ppilysis</json:string><json:string>mutation</json:string><json:string>tam</json:string><json:string>nucleotide</json:string><json:string>azttp</json:string><json:string>aztmp removal</json:string><json:string>chem</json:string><json:string>dntp</json:string><json:string>biol</json:string><json:string>terminator</json:string><json:string>d4tmp removal</json:string><json:string>analogue</json:string><json:string>datum</json:string><json:string>physiological 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curve</json:string><json:string>resistance mutation</json:string><json:string>nucleotide removal</json:string><json:string>synthetic trna3lys</json:string><json:string>larder</json:string><json:string>kinetic constant</json:string><json:string>rate constant</json:string><json:string>dnmp incorporation</json:string><json:string>noncompetitive inhibition</json:string><json:string>model initiation system</json:string><json:string>physiological role</json:string><json:string>more prone</json:string><json:string>different concentration</json:string><json:string>chemical step</json:string><json:string>less sensitive</json:string><json:string>dnmp removal</json:string><json:string>nucleotide analogue</json:string><json:string>high concentration</json:string><json:string>chain terminator removal</json:string><json:string>product formation</json:string><json:string>more sensitive</json:string><json:string>inhibition</json:string></teeft></keywords><author><json:item><name>RAY Adrian S.</name><affiliations><json:string>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</json:string><json:string>Yale University School of Medicine.</json:string></affiliations></json:item><json:item><name>MURAKAMI Eisuke</name><affiliations><json:string>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</json:string><json:string>Yale University School of Medicine.</json:string></affiliations></json:item><json:item><name>BASAVAPATHRUNI Aravind</name><affiliations><json:string>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</json:string><json:string>Yale University School of Medicine.</json:string></affiliations></json:item><json:item><name>VACCARO Joseph A.</name><affiliations><json:string>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</json:string><json:string>Yale University School of Medicine.</json:string><json:string>Current address: Department of Biochemistry, Tulane UniversityHealth Sciences Center, 1430 Tulane Ave. SL-43, New Orleans, LA70112.</json:string></affiliations></json:item><json:item><name>ULRICH Dagny</name><affiliations><json:string>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</json:string><json:string>Yale University School of Medicine.</json:string></affiliations></json:item><json:item><name>CHU Chung K.</name><affiliations><json:string>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</json:string><json:string>The University of Georgia.</json:string></affiliations></json:item><json:item><name>SCHINAZI Raymond F.</name><affiliations><json:string>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</json:string><json:string>Emory University School of Medicine/Veterans Affairs MedicalCenter.</json:string></affiliations></json:item><json:item><name>ANDERSON Karen S.</name><affiliations><json:string>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</json:string><json:string>Yale University School of Medicine.</json:string><json:string>To whom correspondence should be addressed. E-mail: karen.anderson@yale.edu. Phone: (203) 785-4526. Fax: (203) 785-7670.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-1F3PKRB5-X</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase (RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision, suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain terminator from an RNA−RNA primer−template may show how slight changes in selectivity against AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance. Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations. Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.</abstract><qualityIndicators><score>10</score><pdfWordCount>8084</pdfWordCount><pdfCharCount>49928</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>735</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>255</abstractWordCount><abstractCharCount>1815</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1 Reverse Transcriptase Using a Transient Kinetic Analysis†</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>42</volume><issue>29</issue><pages><first>8831</first><last>8841</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-1F3PKRB5-X</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences medicales</json:string></inist></categories><publicationDate>2003</publicationDate><copyrightDate>2003</copyrightDate><doi><json:string>10.1021/bi034435l</json:string></doi><id>27E0B499B0C8B79DCFA4C54E465DD77F7037A1FA</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-1F3PKRB5-X/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-1F3PKRB5-X/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-1F3PKRB5-X/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-1F3PKRB5-X/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1
Reverse Transcriptase Using a Transient Kinetic Analysis<ref type="bib" target="#bi034435lAF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2003 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2003</date><date type="Copyright" when="2003">2003</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1
Reverse Transcriptase Using a Transient Kinetic Analysis<ref type="bib" target="#bi034435lAF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Ray</surname><forename type="first">Adrian S.</forename></persName><affiliation><orgName type="department">Department of Pharmacology</orgName><orgName type="institution">Yale University School of Medicine</orgName><address><addrLine>333 Cedar Street</addrLine><addrLine>New Haven</addrLine><addrLine>Connecticut 06520</addrLine><addrLine>Department of Pharmaceutical and Biomedical Sciences</addrLine><addrLine>College of Pharmacy</addrLine><addrLine>The University of Georgia</addrLine><addrLine>Athens</addrLine><addrLine>Georgia 30602-2352</addrLine><addrLine>and Laboratory of Biochemical Pharmacology</addrLine><addrLine>Department of Pediatrics</addrLine><addrLine>Emory University School of MedicineVeterans Affairs Medical Center</addrLine><addrLine>Decatur</addrLine><addrLine>Georgia 30033</addrLine></address></affiliation><note place="foot" n="bi034435lAF3"><ref>‡</ref><p>
Yale University School of Medicine.</p></note></author><author xml:id="author-0001"><persName><surname>Murakami</surname><forename type="first">Eisuke</forename></persName><affiliation><orgName type="department">Department of Pharmacology</orgName><orgName type="institution">Yale University School of Medicine</orgName><address><addrLine>333 Cedar Street</addrLine><addrLine>New Haven</addrLine><addrLine>Connecticut 06520</addrLine><addrLine>Department of Pharmaceutical and Biomedical Sciences</addrLine><addrLine>College of Pharmacy</addrLine><addrLine>The University of Georgia</addrLine><addrLine>Athens</addrLine><addrLine>Georgia 30602-2352</addrLine><addrLine>and Laboratory of Biochemical Pharmacology</addrLine><addrLine>Department of Pediatrics</addrLine><addrLine>Emory University School of MedicineVeterans Affairs Medical Center</addrLine><addrLine>Decatur</addrLine><addrLine>Georgia 30033</addrLine></address></affiliation><note place="foot" n="bi034435lAF3"><ref>‡</ref><p>
Yale University School of Medicine.</p></note></author><author xml:id="author-0002"><persName><surname>Basavapathruni</surname><forename type="first">Aravind</forename></persName><affiliation><orgName type="department">Department of Pharmacology</orgName><orgName type="institution">Yale University School of Medicine</orgName><address><addrLine>333 Cedar Street</addrLine><addrLine>New Haven</addrLine><addrLine>Connecticut 06520</addrLine><addrLine>Department of Pharmaceutical and Biomedical Sciences</addrLine><addrLine>College of Pharmacy</addrLine><addrLine>The University of Georgia</addrLine><addrLine>Athens</addrLine><addrLine>Georgia 30602-2352</addrLine><addrLine>and Laboratory of Biochemical Pharmacology</addrLine><addrLine>Department of Pediatrics</addrLine><addrLine>Emory University School of MedicineVeterans Affairs Medical Center</addrLine><addrLine>Decatur</addrLine><addrLine>Georgia 30033</addrLine></address></affiliation><note place="foot" n="bi034435lAF3"><ref>‡</ref><p>
Yale University School of Medicine.</p></note></author><author xml:id="author-0003"><persName><surname>Vaccaro</surname><forename type="first">Joseph A.</forename></persName><note place="foot" n="bi034435lAF3"><ref>‡</ref><p>
Yale University School of Medicine.</p></note><note place="foot" n="bi034435lAF4"><ref>§</ref><p>
Current address: Department of Biochemistry, Tulane University
Health Sciences Center, 1430 Tulane Ave. SL-43, New Orleans, LA
70112.</p></note></author><author xml:id="author-0004"><persName><surname>Ulrich</surname><forename type="first">Dagny</forename></persName><affiliation><orgName type="department">Department of Pharmacology</orgName><orgName type="institution">Yale University School of Medicine</orgName><address><addrLine>333 Cedar Street</addrLine><addrLine>New Haven</addrLine><addrLine>Connecticut 06520</addrLine><addrLine>Department of Pharmaceutical and Biomedical Sciences</addrLine><addrLine>College of Pharmacy</addrLine><addrLine>The University of Georgia</addrLine><addrLine>Athens</addrLine><addrLine>Georgia 30602-2352</addrLine><addrLine>and Laboratory of Biochemical Pharmacology</addrLine><addrLine>Department of Pediatrics</addrLine><addrLine>Emory University School of MedicineVeterans Affairs Medical Center</addrLine><addrLine>Decatur</addrLine><addrLine>Georgia 30033</addrLine></address></affiliation><note place="foot" n="bi034435lAF3"><ref>‡</ref><p>
Yale University School of Medicine.</p></note></author><author xml:id="author-0005"><persName><surname>Chu</surname><forename type="first">Chung K.</forename></persName><affiliation><orgName type="department">Department of Pharmacology</orgName><orgName type="institution">Yale University School of Medicine</orgName><address><addrLine>333 Cedar Street</addrLine><addrLine>New Haven</addrLine><addrLine>Connecticut 06520</addrLine><addrLine>Department of Pharmaceutical and Biomedical Sciences</addrLine><addrLine>College of Pharmacy</addrLine><addrLine>The University of Georgia</addrLine><addrLine>Athens</addrLine><addrLine>Georgia 30602-2352</addrLine><addrLine>and Laboratory of Biochemical Pharmacology</addrLine><addrLine>Department of Pediatrics</addrLine><addrLine>Emory University School of MedicineVeterans Affairs Medical Center</addrLine><addrLine>Decatur</addrLine><addrLine>Georgia 30033</addrLine></address></affiliation><note place="foot" n="bi034435lAF5"><ref>‖</ref><p>
The University of Georgia.</p></note></author><author xml:id="author-0006"><persName><surname>Schinazi</surname><forename type="first">Raymond F.</forename></persName><affiliation><orgName type="department">Department of Pharmacology</orgName><orgName type="institution">Yale University School of Medicine</orgName><address><addrLine>333 Cedar Street</addrLine><addrLine>New Haven</addrLine><addrLine>Connecticut 06520</addrLine><addrLine>Department of Pharmaceutical and Biomedical Sciences</addrLine><addrLine>College of Pharmacy</addrLine><addrLine>The University of Georgia</addrLine><addrLine>Athens</addrLine><addrLine>Georgia 30602-2352</addrLine><addrLine>and Laboratory of Biochemical Pharmacology</addrLine><addrLine>Department of Pediatrics</addrLine><addrLine>Emory University School of MedicineVeterans Affairs Medical Center</addrLine><addrLine>Decatur</addrLine><addrLine>Georgia 30033</addrLine></address></affiliation><note place="foot" n="bi034435lAF6"><ref>⊥</ref><p>
Emory University School of Medicine/Veterans Affairs Medical
Center.</p></note></author><author xml:id="author-0007" role="corresp"><persName><surname>Anderson</surname><forename type="first">Karen S.</forename></persName><note place="foot" n="bi034435lAF3"><ref>‡</ref><p>
Yale University School of Medicine.</p></note><affiliation role="corresp"> To whom correspondence should be addressed. E-mail: karen.anderson@yale.edu. Phone: (203) 785-4526. Fax: (203) 785-7670.</affiliation></author><idno type="istex">27E0B499B0C8B79DCFA4C54E465DD77F7037A1FA</idno><idno type="ark">ark:/67375/TPS-1F3PKRB5-X</idno><idno type="DOI">10.1021/bi034435l</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2003</date><date type="published">2003</date><biblScope unit="vol">42</biblScope><biblScope unit="issue">29</biblScope><biblScope unit="page" from="8831">8831</biblScope><biblScope unit="page" to="8841">8841</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase
(RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside
resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of
AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural
deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference
between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide
removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision,
suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated
removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance
mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide
during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant
can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain
terminator from an RNA−RNA primer−template may show how slight changes in selectivity against
AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance.
Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations.
Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions
with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi034435l</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1
Reverse Transcriptase Using a Transient Kinetic Analysis<xref rid="bi034435lAF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Ray</surname><given-names>Adrian S.</given-names></name><xref rid="bi034435lAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Murakami</surname><given-names>Eisuke</given-names></name><xref rid="bi034435lAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Basavapathruni</surname><given-names>Aravind</given-names></name><xref rid="bi034435lAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Vaccaro</surname><given-names>Joseph A.</given-names></name><xref rid="bi034435lAF3"><sup>‡</sup></xref><xref rid="bi034435lAF4"><sup>§</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Ulrich</surname><given-names>Dagny</given-names></name><xref rid="bi034435lAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Chu</surname><given-names>Chung K.</given-names></name><xref rid="bi034435lAF5"><sup>‖</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Schinazi</surname><given-names>Raymond F.</given-names></name><xref rid="bi034435lAF6"><sup>⊥</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Anderson</surname><given-names>Karen S.</given-names></name><xref rid="bi034435lAF1">*</xref><xref rid="bi034435lAF3"><sup>‡</sup></xref></contrib><aff>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,
Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,
Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033
</aff></contrib-group><author-notes><fn id="bi034435lAF3"><label>‡</label><p>
Yale University School of Medicine.</p></fn><fn id="bi034435lAF4"><label>§</label><p>
Current address: Department of Biochemistry, Tulane University
Health Sciences Center, 1430 Tulane Ave. SL-43, New Orleans, LA
70112.</p></fn><fn id="bi034435lAF5"><label>‖</label><p>
The University of Georgia.</p></fn><fn id="bi034435lAF6"><label>⊥</label><p>
Emory University School of Medicine/Veterans Affairs Medical
Center.</p></fn><corresp id="bi034435lAF1">
To whom correspondence should be addressed. E-mail:
karen.anderson@yale.edu. Phone: (203) 785-4526. Fax: (203) 785-7670.</corresp></author-notes><pub-date pub-type="epub"><day>4</day><month>07</month><year>2003</year></pub-date><pub-date pub-type="ppub"><day>29</day><month>07</month><year>2003</year></pub-date><volume>42</volume><issue>29</issue><fpage>8831</fpage><lpage>8841</lpage><history><date date-type="received"><day>18</day><month>03</month><year>2003</year></date><date date-type="rev-recd"><day>20</day><month>05</month><year>2003</year></date><date date-type="asap"><day>4</day><month>07</month><year>2003</year></date><date date-type="issue-pub"><day>29</day><month>07</month><year>2003</year></date></history><permissions><copyright-statement>Copyright © 2003 American Chemical Society</copyright-statement><copyright-year>2003</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase
(RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside
resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of
AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural
deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference
between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide
removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision,
suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated
removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance
mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide
during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant
can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain
terminator from an RNA−RNA primer−template may show how slight changes in selectivity against
AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance.
Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations.
Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions
with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi034435l</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi034435lAF2"><label>†</label><p>
Work supported by NIH Grants GM49551 (K.S.A.), R37AI-41980
(R.F.S.), AI 25899 (C.K.C.), and RO1AI-32351 (R.F.S. and C.K.C.),
the Department of Veteran Affairs (R.F.S.), NRSA 5 T32 GM07223
from the National Institute of General Medical Sciences (A.S.R.), and
ACS Postdoctoral Fellowship PF-4478 (J.A.V.).</p></notes></front><body><sec id="d7e210"><title></title><p>Human immunodeficiency virus (HIV), the etiological
agent of acquired immunodeficiency syndrome (AIDS),
requires reverse transcriptase (RT)<sup>1</sup> to copy its single-stranded RNA genome into a double-stranded DNA copy
for integration into the host cell genome during the early
stages of viral infection. Although almost all aspects of the
HIV-1 life cycle have been targeted (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00002" ref-type="bibr"></xref>−<xref rid="bi034435lb00003" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00004" ref-type="bibr"></xref></named-content></italic>), a majority of
the drugs that have been effective are nucleoside reverse
transcriptase inhibitors (NRTIs) which lack a 3‘-hydroxyl
group and serve to chain-terminate viral transcripts. However,
treatment with NRTIs is limited by their toxicity to the host
[often through their interaction with mitochondrial DNA
polymerase γ (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00005" ref-type="bibr"></xref>−<xref rid="bi034435lb00006" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00007" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00008" ref-type="bibr"></xref></named-content></italic>)] and the ability of the virus to mutate
and acquire resistance (<italic toggle="yes"><xref rid="bi034435lb00009" ref-type="bibr"></xref></italic>). Other factors that govern the
efficacy of these inhibitors include uptake, transport, metabolism, and incorporation of the drugs (<italic toggle="yes"><xref rid="bi034435lb00010" ref-type="bibr"></xref></italic>).
</p><p>Several hypotheses have been put forth to explain the
decreased susceptibility of HIV to AZT following prolonged
treatment. Some of these theories stem from cellular factors,
including kinases (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00011" ref-type="bibr"></xref>−<xref rid="bi034435lb00012" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00013" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00014" ref-type="bibr"></xref></named-content></italic>), transporters (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00015" ref-type="bibr"></xref>, <xref rid="bi034435lb00016" ref-type="bibr"></xref></named-content></italic>), and exonucleases (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00017" ref-type="bibr"></xref>, <xref rid="bi034435lb00018" ref-type="bibr"></xref></named-content></italic>). While these factors potentially play
important roles in drug sensitivity, high-level AZT resistance
most likely resides within the virally encoded RT. The high
rate of HIV replication and the lack of proofreading by RT
during reverse transcription lead to frequent mutations (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00019" ref-type="bibr"></xref>−<xref rid="bi034435lb00020" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00021" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00022" ref-type="bibr"></xref></named-content></italic>). One class of mutations includes those originally found
to confer resistance to AZT (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00023" ref-type="bibr"></xref>, <xref rid="bi034435lb00024" ref-type="bibr"></xref></named-content></italic>). Later findings showed
that these mutations also decreased sensitivity to D4T in
patients (<italic toggle="yes"><xref rid="bi034435lb00013" ref-type="bibr"></xref></italic>) despite little or no cross resistance in cell culture
(<italic toggle="yes"><xref rid="bi034435lb00025" ref-type="bibr"></xref></italic>). Their association with thymidine analogues led them
to be termed thymidine-associated mutations (TAMs). Common TAMs include M41L, D67N, K70R, L210W, T215Y/F, and K219Q (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00023" ref-type="bibr"></xref>, <xref rid="bi034435lb00026" ref-type="bibr"></xref></named-content></italic>). Reports have suggested these
mutations cause resistance through slight decreases in the
level of AZTMP incorporation during RNA-directed processes (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00027" ref-type="bibr"></xref>, <xref rid="bi034435lb00028" ref-type="bibr"></xref></named-content></italic>) and an increase in the extent of chain
terminator removal by ATP (<italic toggle="yes"><xref rid="bi034435lb00029" ref-type="bibr"></xref></italic>) or pyrophosphate (PP<sub>i</sub>) (<italic toggle="yes"><xref rid="bi034435lb00030" ref-type="bibr"></xref></italic>).
Removal reactions release the chain terminator as either a
nucleoside triphosphate or a nucleoside tetraphosphate linked
to adenosine for PP<sub>i</sub>- or ATP-mediated removal reactions,
respectively (<italic toggle="yes"><xref rid="bi034435lb00029" ref-type="bibr"></xref></italic>). Although each of these hypotheses superficially explains AZT resistance, there has been much
discussion about their individual contributions to resistance
observed during clinical treatment.
</p><p>Adding more complexity to the understanding of TAMs
is the finding that the presence of various combinations of
these mutations correlates with the reduced activity of non-thymidine analogues, including abacavir, a prodrug of the
guanosine analogue carbovir (CBV) (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00031" ref-type="bibr"></xref>, <xref rid="bi034435lb00032" ref-type="bibr"></xref></named-content></italic>), and PMPA,
an acyclic adenosine monophosphate analogue (<italic toggle="yes"><xref rid="bi034435lb00033" ref-type="bibr"></xref></italic>). In fact,
characteristic TAMs have been found in the resistance
mutation spectra for all NRTIs currently available in the
clinic. Therefore, TAMs appear to be involved in multidrug
resistance, and the prevalence of these mutations poses a
serious threat to the efficacy of nucleosides already approved
by the Food and Drug Administration as well as those being
developed as future treatments.
</p><p>This report is a quantitative exploration of the underlying
mechanisms that govern the resistance of TAM-containing
RT to AZT and other compounds. Using a pre-steady-state
kinetic approach, we studied PP<sub>i</sub>- and ATP-mediated deoxynucleotide removal as well as AZTMP incorporation into a
model initiation complex containing synthetic tRNA<sub>3</sub><sup>Lys</sup>.
Observations made during these studies suggest that ATP-
and PP<sub>i</sub>-mediated deoxynucleotide removal, as well as
increased selectivity against AZTMP incorporation during
RNA-directed processes, may all contribute to high-level
resistance to AZT. Studies aimed at understanding how
structural features of the deoxynucleotide analogue affect
removal suggest that analogues with the potential to form
strong interactions in the RT active site may be more prone
to excision.
</p></sec><sec id="d7e316"><title>Materials and Methods</title><p><italic toggle="yes">Preparation of HIV-1 RT. </italic>RT<sup>WT</sup> and RT<sup>AZTR</sup> clones were
generously provided by S. Hughes, P. Boyer, and A. Ferris
(Frederick Cancer Research and Development Center, Frederick, MD). The N-terminal histidine-tagged heterodimeric
p66 and p51 enzymes were purified as previously described
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00034" ref-type="bibr"></xref>, <xref rid="bi034435lb00035" ref-type="bibr"></xref></named-content></italic>). Protein active site concentrations were determined
using pre-steady-state burst experiments where the primer−template (300 nM) is in slight excess over RT (100 nM total)
and the amplitude of the initial exponential phase of product
formation is correlated with the amount of active protein in
the experiment (<italic toggle="yes"><xref rid="bi034435lb00036" ref-type="bibr"></xref></italic>). This methodology allowed for the
accurate normalization of protein concentrations of different
mutants and protein preparations. It is imperative that an
accurate determination of protein active site concentration
be carried out to allow for meaningful comparisons between
wild-type and mutant RT.
</p><p><italic toggle="yes">Nucleoside Triphosphates.</italic> Natural 2‘-deoxynucleotides
were purchased from Amersham. AZTTP and D4TTP were
obtained from Moravek Biochemicals.
</p><p><italic toggle="yes">Oligonucleotides.</italic> Primers and templates used for incorporation and removal studies are shown in Table <xref rid="bi034435lt00001"></xref>, and all
of the DNA oligos were synthesized on an Applied Biosystems DNA synthesizer (Keck DNA synthesis facility, Yale
University, New Haven, CT) and purified using 20%
polyacrylamide denaturing gel electrophoresis. The R18-,
R36-, and R45-mers were synthesized and purified by New
England Biolabs. Chain-terminated oligos were made by
incorporation with RT by previously reported methods (<italic toggle="yes"><xref rid="bi034435lb00007" ref-type="bibr"></xref></italic>).
DNA and RNA primers were 5‘-<sup>32</sup>P-labeled with T4 polynucleotide kinase (New England Biolabs) as previously
described (<italic toggle="yes"><xref rid="bi034435lb00036" ref-type="bibr"></xref></italic>). [γ-<sup>32</sup>P]ATP was purchased from Amersham.
Biospin columns for the removal of excess [γ-<sup>32</sup>P]ATP were
purchased from Bio-Rad. Synthetic tRNA<sub>3</sub><sup>Lys</sup> was T7 transcribed from cut plasmid kindly provided by J. Pata and T.
A. Steitz (Yale University). Transcription was followed by
5‘- and 3‘-hammerhead ribozyme cleavage to create homogeneous ends by methods previously described (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00037" ref-type="bibr"></xref>, <xref rid="bi034435lb00038" ref-type="bibr"></xref></named-content></italic>).
Synthetic tRNA<sub>3</sub><sup>Lys</sup> was labeled with <sup>32</sup>P by internal labeling
during transcription by the addition of 250 μCi of each
α-labeled NTP (1 mCi total). [α-<sup>32</sup>P]ATP, [α-<sup>32</sup>P]GTP,
[α-<sup>32</sup>P]CTP, and [α-<sup>32</sup>P]UTP were purchased from Amersham.
<table-wrap id="bi034435lt00001" position="float" orientation="portrait"><label>1</label><caption><p>Sequences of Primers and Templates Used To Study Incorporation and Removal</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"><graphic xlink:href="bi034435lu00001a.tif" position="float" orientation="portrait"></graphic></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> DNA 36-mer has the same sequence with 2‘-deoxyribonucleotides
and U's changed to T's.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Z is AZTMP.<italic toggle="yes"><sup>c</sup></italic><sup></sup> X is AZTMP, D4TMP, dCMP
(D23-mer), ddCMP, 3TCMP, or D4CMP.<italic toggle="yes"><sup>d</sup></italic><sup></sup> Y is dCMP (D24-mer) or
dCMPαS.<italic toggle="yes"><sup>e</sup></italic><sup></sup> N is A (D45). N is G (D45C).</p></table-wrap-foot></table-wrap></p><p>Annealing of the DNA and RNA primer−templates was
carried out by adding a 1:1.4 molar ratio of purified primer
to template at 90 °C for 5 min, 50 °C for 10 min, and 0 °C
for 10 min. The annealed primer and template were then
analyzed using 15% nondenaturing polyacrylamide gel
electrophoresis to ensure complete annealing. Concentrations
of the oligonucleotides were estimated by UV absorbance
at 260 nm using calculated extinction coefficients.
</p><p><italic toggle="yes">Pre-Steady-State Removal Assays</italic>. ATP- and PP<sub>i</sub>-mediated
removal were studied as previously described (<italic toggle="yes"><xref rid="bi034435lb00039" ref-type="bibr"></xref></italic>) by
measuring the decrease in the length of the primer either at
a physiological concentration of PP<sub>i</sub> (125 μM) or ATP (3
mM) or by determining the dependence of concentration on
rate by titrating the removing agent. Pyrophosphate exchange
experiments for studying the removal of AZTMP were
performed as described previously (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00040" ref-type="bibr"></xref>, <xref rid="bi034435lb00041" ref-type="bibr"></xref></named-content></italic>) by assessing the
exchange of <sup>32</sup>P-labeled PP<sub>i</sub> into an unlabeled pool of AZTTP
during the process of incorporation and removal by HIV-1
RT. The reaction mixture contained 250 nM D22−D45, 20
μM AZTTP, 10 mM MgCl<sub>2</sub>, 500 nM RT, and varying
concentrations of <sup>32</sup>P-labeled PP<sub>i</sub>. Formation of the labeled
AZTTP was assessed by thin-layer chromatography using
PEI-cellulose plates and 0.3 M potassium phosphate buffer
(pH 8.0) as a mobile phase. The products were visualized
and quantitated by phosphorimaging on a Bio-Rad GS-525
Molecular Imager System.
</p><p>The inhibition constant (<italic toggle="yes">K</italic><sub>i</sub>) for the next correct nucleotide
on removal was determined by examining PP<sub>i</sub> binding in
the presence of different concentrations of the next correct
nucleotide (dCTP). Two approaches were used to determine
the concentration necessary to inhibit ATP-mediated removal
by 50% (IC<sub>50</sub>): (i) the decrease in primer length measured
directly (as described above) or (ii) a “limited rescue”
methodology (described below). Comparison of the rates
obtained from the two methodologies revealed similar results
(data not shown). For limited rescue, 10 μM TTP was added
as well as varying concentrations of the next correct
nucleotide. A physiological concentration of ATP (3 mM)
was used to stimulate removal. The ATP was pretreated with
pyrophosphatase to ensure that there was no contaminating
PP<sub>i</sub> that would complicate interpretation of results. These
experiments allowed for the quantitation of dNMP removal
by assessing the elongation of the primer. A distinct
advantage of the limited rescue methodology over direct
measurement of removal by decreased primer length was
that the reactions were found to go to completion, allowing
for more precise quantitation. This method also eliminated
possible interference from the presence of all four dNTPs
and/or the addition of a separate polymerase for elongation.
The IC<sub>50</sub> was obtained by plotting the percent inhibition
versus the concentration of next correct nucleotide and fitting
the data to hyperbolic curves.
</p><p><italic toggle="yes">Pre-Steady-State Single-Turnover Incorporation Experiments.</italic> Rapid chemical quench experiments were performed
as previously described with a KinTek Instruments model
RQF-3 rapid-quench-flow apparatus (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00034" ref-type="bibr"></xref>, <xref rid="bi034435lb00036" ref-type="bibr"></xref></named-content></italic>). A pre-steady-state kinetic analysis was used to examine incorporation. The
reactions were carried out by rapidly mixing a solution
containing the preincubated complex of 250 nM HIV-1 RT
(active site concentration) and 50 nM 5‘-labeled duplex with
a solution of 10 mM MgCl<sub>2</sub> and varying concentrations of
the next correct dNTP in the presence of 50 mM Tris-HCl
and 50 mM NaCl at pH 7.8 and 37 °C (all concentrations
represent the final concentration after mixing). Single-turnover experiments were also used to study incorporation
into the synthetic tRNA<sub>3</sub><sup>Lys</sup> as previously described (<italic toggle="yes"><xref rid="bi034435lb00027" ref-type="bibr"></xref></italic>). This
required the addition of both dCTP (2 mM) and varying
concentrations of TTP or AZTTP. Control experiments
showed dCMP incorporation not to be rate-limiting (data not
shown). Products and reactants were then separated on a 10%
polyacrylamide gel containing 7 M urea and 40% formamide
and visualized and quantitated using phosphorimaging.
</p><p><italic toggle="yes">Data Analysis.</italic> Data were fit by nonlinear regression using
the program KaleidaGraph (Synergy Software, Reading, PA).
Single-turnover incorporation and removal experiments were
fit to single-exponential equations: [product] = <italic toggle="yes">A</italic>[1 − exp(−<italic toggle="yes">k</italic><sub>obsd</sub>)] and [product] = <italic toggle="yes">A</italic>[exp(−<italic toggle="yes">k</italic><sub>obsd</sub><italic toggle="yes">t</italic>)], respectively, where
<italic toggle="yes">A</italic> is the amplitude of product formation and <italic toggle="yes">k</italic><sub>obsd</sub> is the
observed rate at a specific substrate concentration. The
dissociation constant (<italic toggle="yes">K</italic><sub>d</sub>) of the substrate (dNTP, PP<sub>i</sub>, or
ATP) binding to the complex of RT and the primer−template
during dNMP incorporation or removal was calculated by
fitting observed rate constants at different concentrations of
dNTP to the hyperbolic equation <italic toggle="yes">k</italic><sub>obsd</sub> = <italic toggle="yes">k</italic><sub>pol</sub>[dNTP]/(<italic toggle="yes">K</italic><sub>d</sub> +
[dNTP]), where <italic toggle="yes">k</italic><sub>pol</sub> is the maximum first-order rate constant
for dNMP incorporation and <italic toggle="yes">K</italic><sub>d</sub> is the equilibrium dissociation constant for the productive interaction of dNTP with
the E·DNA complex. PP<sub>i</sub> exchange rates were calculated by
the previously derived equation (<italic toggle="yes"><xref rid="bi034435lb00042" ref-type="bibr"></xref></italic>) <italic toggle="yes">k</italic><sub>exchange</sub> = {−[[PP<sub>i</sub>][AZTTP]/([PP<sub>i</sub>] + [AZTTP])](1/<italic toggle="yes">T</italic>) ln(1 − <italic toggle="yes">F</italic>)}/[complex],
where <italic toggle="yes">F</italic> is the fraction of isotopic equilibrium at time <italic toggle="yes">T</italic> and
[complex] is the concentration of enzyme-bound DNA (250
nM). A slope of the linear region from the plot of ln(1 − <italic toggle="yes">F</italic>)
versus <italic toggle="yes">T</italic> is a function of the rate of isotope exchange.
</p><p>To determine the mechanism of inhibition of PP<sub>i</sub>-mediated
removal by the next correct nucleotide, hyperbolic curves
were fit to the rate of PP<sub>i</sub> lysis removal versus concentration
for the data generated in the absence or presence of
increasing concentrations of the next correct nucleotide. A
significant increase in the <italic toggle="yes">K</italic><sub>d</sub> or decrease in the <italic toggle="yes">k</italic><sub>pol</sub> indicated
competitive or noncompetitive mechanisms of inhibition,
respectively. Consistency was observed for the mechanism
of inhibition at differing concentrations of the next correct
nucleotide for a specific RT enzyme and chain-terminated
primer−template substrate. <italic toggle="yes">K</italic><sub>i</sub> values were determined by
fitting the data generated in the presence of inhibitor to the
hyperbolic binding equation for competitive {<italic toggle="yes">k</italic><sub>obsd</sub> = (<italic toggle="yes">k</italic><sub>pol</sub>[dNTP])/[[dNTP] + <italic toggle="yes">K</italic><sub>d</sub>(1 + [I]/<italic toggle="yes">K</italic><sub>i</sub><sup>com</sup>)]}, noncompetitive
{<italic toggle="yes">k</italic><sub>obsd</sub> = [(<italic toggle="yes">k</italic><sub>pol</sub>[dNTP])/(1 + [I]/<italic toggle="yes">K</italic><sub>i</sub><sup>non</sup>)]/(<italic toggle="yes">K</italic><sub>d</sub> + [dNTP])}, or
mixed inhibition {<italic toggle="yes">k</italic><sub>obsd</sub> = [(<italic toggle="yes">k</italic><sub>pol</sub>[dNTP])/(1 + [I]/<italic toggle="yes">K</italic><sub>i</sub><sup>non</sup>)]/[[dNTP] + <italic toggle="yes">K</italic><sub>d</sub>(1 + [I]/<italic toggle="yes">K</italic><sub>i</sub><sup>com</sup>)]}. Brackets denote concentration, and [I] equals the concentration of inhibitor at which
the binding curve was determined. Reported errors represent
the deviation of points from the curve fit generated by
KaleidaGraph or were calculated by standard statistical
analysis (<italic toggle="yes"><xref rid="bi034435lb00043" ref-type="bibr"></xref></italic>).
</p></sec><sec id="d7e720"><title>Results</title><p><italic toggle="yes">Mechanism of Nucleotide Removal by HIV-1 RT. </italic>The first
step in understanding AZT resistance is to fully define the
mechanism of nucleotide incorporation and/or removal by
RT. Previous reports provide information about the forward
and reverse reactions catalyzed by HIV-1 RT (<italic toggle="yes">36</italic>,<italic toggle="yes"> 44−46</italic>).
The small changes with respect to incorporation for AZT
resistant RT (RT<sup>AZTR</sup>) (<italic toggle="yes">27</italic>,<italic toggle="yes"> 28</italic>,<italic toggle="yes"> 47</italic>,<italic toggle="yes"> 48</italic>) further focus attention
on the reverse reaction as a possible means of resistance.
One aspect of the mechanism of removal that has not been
thoroughly addressed is whether a rate-limiting step precedes
or occurs after pyrophosphate (PP<sub>i</sub>) release after the incorporation of deoxynucleotide (Scheme <xref rid="bi034435lh00001"></xref>). Model 2 is potentially interesting because PP<sub>i</sub> binding to the RT·primer−template complex in an activated form may allow for more
rapid removal. This faster step could be masked by earlier
slower steps in experiments that were done with pre-chain-terminated primer−templates, yielding data irrelevant to the
physiologically pertinent removal reaction.
<fig id="bi034435lh00001" position="float" fig-type="scheme" orientation="portrait"><label>1</label><caption><p>Models for the Kinetic Mechanism of PP<sub>i</sub> Release after dNMP Incorporation by HIV-1 RT
</p></caption><graphic xlink:href="bi034435lh00001.tif" position="float" orientation="portrait"></graphic></fig></p><p>A comparison of the rates of pyrophosphorolysis (PP<sub>i</sub>lysis)
and PP<sub>i</sub> exchange has proven to be useful in addressing the
mechanism of the reverse reaction for other DNA polymerases (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00040" ref-type="bibr"></xref>, <xref rid="bi034435lb00041" ref-type="bibr"></xref></named-content></italic>). To distinguish between the two models,
the PP<sub>i</sub>lysis rate for AZTMP removal was compared to the
rate of exchange of <sup>32</sup>PP<sub>i</sub> with unlabeled AZTTP during
rounds of both incorporation and removal with D22−D45
(primers and templates shown in Table <xref rid="bi034435lt00001"></xref>). The similarity in
rates of PP<sub>i</sub>lysis and PP<sub>i</sub> exchange (Table <xref rid="bi034435lt00002"></xref>) suggests that,
as for T7 DNA polymerase (<italic toggle="yes"><xref rid="bi034435lb00040" ref-type="bibr"></xref></italic>) and DNA polymerase I (<italic toggle="yes"><xref rid="bi034435lb00041" ref-type="bibr"></xref></italic>),
the rate-limiting step occurs before PP<sub>i</sub> release, suggesting
that model 1 correctly reflects the mechanism followed by
RT<sup>WT</sup>. To determine if mutations in RT<sup>AZTR</sup> caused any
changes in the rate of PP<sub>i</sub>lysis or PP<sub>i</sub> exchange, similar
experiments were carried out with the mutant enzyme. Rates
of PP<sub>i</sub> exchange by RT<sup>WT</sup> and RT<sup>AZTR</sup> were found to be
similar (Figure <xref rid="bi034435lf00001"></xref>A−C). These results further suggest that
studies looking directly at the removal reaction from chain-terminated primers used throughout this paper were valid.
<fig id="bi034435lf00001" position="float" orientation="portrait"><label>1</label><caption><p>Determination of the PP<sub>i</sub> exchange rate. (A) Products
from a PP<sub>i</sub> exchange reaction in which <sup>32</sup>P-labeled PP<sub>i</sub> was
exchanged into an unlabeled pool of AZTTP during rounds of
incorporation and removal were separated using thin-layer chromatography. The positions of PP<sub>i</sub>, AZTTP, and contaminating P<sub>i</sub>
are labeled. (B) Rate of PP<sub>i</sub> exchange for RT<sup>WT</sup> (·) and RT<sup>AZTR</sup>
(○) in the presence of 125 μM PP<sub>i</sub>. The rates of exchange (<italic toggle="yes">k</italic><sub>ex</sub>)
were 0.014 ± 0.001 and 0.015 ± 0.001 s<sup>-1</sup> for RT<sup>WT</sup> and RT<sup>AZTR</sup>,
respectively. (C) PP<sub>i</sub> exchange in the presence of 1 mM PP<sub>i</sub>. <italic toggle="yes">k</italic><sub>ex</sub>
equaled 0.073 ± 0.002 and 0.063 ± 0.001 s<sup>-1</sup> for RT<sup>WT</sup> and
RT<sup>AZTR</sup>, respectively.</p></caption><graphic xlink:href="bi034435lf00001.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi034435lt00002" position="float" orientation="portrait"><label>2</label><caption><p>Phosphorothioate Effect for the Forward versus Reverse Reaction and PP<sub>i</sub>lysis versus PP<sub>i</sub> Exchange by RT<sup>WT</sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="2"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">reaction</oasis:entry><oasis:entry namest="2" nameend="2">forward <italic toggle="yes">k</italic><sub>obsd</sub> (s<sup>-1</sup>)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D23 + dCMP
</oasis:entry><oasis:entry colname="2">1.7 ± 0.1
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D23 + dCMPαS
</oasis:entry><oasis:entry colname="2">1.3 ± 0.1</oasis:entry></oasis:row><oasis:row><oasis:entry namest="1" nameend="1">reaction</oasis:entry><oasis:entry namest="2" nameend="2">reverse initial velocity (nM/s<sup>-1</sup>)<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D24 − dCMP
</oasis:entry><oasis:entry colname="2">0.28
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D24 − dCMPαS
</oasis:entry><oasis:entry colname="2">0.043</oasis:entry></oasis:row><oasis:row><oasis:entry namest="1" nameend="1">reaction</oasis:entry><oasis:entry namest="2" nameend="2"><italic toggle="yes">k</italic><sub>obsd</sub> (s<sup>-1</sup>)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PP<sub>i</sub>lysis<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="2">0.010 ± 0.002
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PP<sub>i</sub> exchange<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="2">0.014 ± 0.001</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> The velocity before the rapid attainment of equilibrium.<italic toggle="yes"><sup>b</sup></italic><sup></sup> PP<sub>i</sub>lysis
and PP<sub>i</sub> exchange reactions were carried out in the presence of 125
μM PP<sub>i</sub>.</p></table-wrap-foot></table-wrap></p><p>Large phosphorothioate elemental effects have previously
been thought to indicate a rate-limiting chemical step (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00040" ref-type="bibr"></xref>,
<xref rid="bi034435lb00049" ref-type="bibr"></xref></named-content></italic>), although results call into question the expected magnitude of the rate change (<italic toggle="yes"><xref rid="bi034435lb00050" ref-type="bibr"></xref></italic>). Experiments were carried out
in an effort to examine the effects of a nonbridging sulfur
atom on single-turnover incorporation and PP<sub>i</sub>lysis in the
presence of dCTPαS and dCMPαS-terminated primer,
respectively. An excess of enzyme and high concentrations
of both enzyme and primer−template were used to limit the
possibility of large phosphorothioate binding effects observed
with other polymerases, further confounding mechanistic
conclusions (<italic toggle="yes"><xref rid="bi034435lb00041" ref-type="bibr"></xref></italic>). Phosphorothioate effects of 1.3- and 6.5-fold were observed during incorporation and removal,
respectively (Table <xref rid="bi034435lt00002"></xref>). These data tentatively suggest differences in the rate-limiting steps for catalysis in the forward
and reverse direction.
</p><p><italic toggle="yes">Equilibrium Binding Constant (K<sub>d</sub></italic><italic toggle="yes">) and Maximum Rate
of Removal (k<sub>rem</sub></italic><italic toggle="yes">) for PP<sub>i</sub></italic>-<italic toggle="yes"> and ATP-Mediated Removal by
RT<sup>WT</sup></italic><sup></sup><italic toggle="yes"> and RT<sup>AZTR</sup></italic><sup></sup><italic toggle="yes"> during Different Stages of HIV-1 Replication. </italic>The PP<sub>i</sub> and ATP concentration dependence on the rate
of removal of a terminal AZTMP from different RNA and
DNA primer−template substrates was determined for RT<sup>WT</sup>
and RT<sup>AZTR</sup> in an effort to further understand the mechanistic
consequences of D67N, K70R, T215Y, and K219Q mutations within the AZT resistant quadruple mutant on removal
(Table <xref rid="bi034435lt00003"></xref> and Figure <xref rid="bi034435lf00002"></xref>). The kinetic data show that RT<sup>AZTR</sup>
is slightly impaired during DNA- and RNA-directed PP<sub>i</sub>-mediated removal compared to RT<sup>WT</sup> (Figure <xref rid="bi034435lf00002"></xref>A,C). Conversely, during ATP-mediated removal from both DNA−DNA and DNA−RNA primer−templates, an as much as 10-fold increase in rate led RT<sup>AZTR</sup> to be more efficient than
RT<sup>WT</sup> (Figure <xref rid="bi034435lf00002"></xref>B,D). During both PP<sub>i</sub>- and ATP-mediated
removal, no AZTMP excision was observed from an RNA−RNA primer−template.
<fig id="bi034435lf00002" position="float" orientation="portrait"><label>2</label><caption><p>Removal of AZTMP from the DNA−DNA primer−template by RT<sup>WT</sup> and RT<sup>AZTR</sup>. (A) Family of curves for the removal of
AZTMP by RT<sup>AZTR</sup> and 0.125 (·), 0.5 (◇), 1 (▵), 2.5 (×), and 6 mM PP<sub>i</sub> (+). (B) Family of curves for the removal of AZTMP by RT<sup>AZTR</sup>
by 0.1 (○), 0.25 (·), 1 (▵), 2.5 (×), and 6 mM ATP (+). (C) Dependence of the first-order rate constant (<italic toggle="yes">k</italic><sub>obsd</sub>) for AZTMP removal on
PP<sub>i</sub> concentration for RT<sup>WT</sup> (·) and RT<sup>AZTR</sup> (○). Data fit to hyperbolic curves giving maximum rates of removal (<italic toggle="yes">k</italic><sub>rem</sub>) of 0.15 ± 0.02 and
0.22 ± 0.03 s<sup>-1</sup> and dissociation constants (<italic toggle="yes">K</italic><sub>d</sub>) of 0.97 ± 0.36 and 4.2 ± 1.2 mM for RT<sup>WT</sup> and RT<sup>AZTR</sup>, respectively. (D) Dependence of
<italic toggle="yes">k</italic><sub>obsd</sub> for AZTMP removal on ATP concentration for RT<sup>WT</sup> (·) and RT<sup>AZTR</sup> (○). <italic toggle="yes">k</italic><sub>rem</sub> values were 0.00056 ± 0.00004 and 0.0028 ± 0.0002
s<sup>-1</sup> and <italic toggle="yes">K</italic><sub>d</sub> values 0.87 ± 0.23 and 0.32 ± 0.10 mM for RT<sup>WT</sup> and RT<sup>AZTR</sup>, respectively.
</p></caption><graphic xlink:href="bi034435lf00002.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi034435lt00003" position="float" orientation="portrait"><label>3</label><caption><p>Kinetic Parameters for AZTMP Removal during Various Stages of Replication</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="7"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:colspec colnum="7" colname="7"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">primer−template</oasis:entry><oasis:entry namest="2" nameend="2">substrate</oasis:entry><oasis:entry namest="3" nameend="3">protein</oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">k</italic><sub>rem</sub> (s<sup>-1</sup>)<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry namest="5" nameend="5"><italic toggle="yes">K</italic><sub>d </sub>(mM)</oasis:entry><oasis:entry namest="6" nameend="6">efficiency<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry namest="7" nameend="7"><italic toggle="yes">k</italic><sub>phys</sub> (s<sup>-1</sup>)<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">DNA−DNA
</oasis:entry><oasis:entry colname="2">PP<sub>i</sub></oasis:entry><oasis:entry colname="3">WT
</oasis:entry><oasis:entry colname="4">0.15 ± 0.02
</oasis:entry><oasis:entry colname="5">0.97 ± 0.36
</oasis:entry><oasis:entry colname="6">0.15
</oasis:entry><oasis:entry colname="7">0.017
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">AZTR
</oasis:entry><oasis:entry colname="4">0.22 ± 0.03
</oasis:entry><oasis:entry colname="5">4.2 ± 1.2
</oasis:entry><oasis:entry colname="6">0.052
</oasis:entry><oasis:entry colname="7">0.0064
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">ATP
</oasis:entry><oasis:entry colname="3">WT
</oasis:entry><oasis:entry colname="4">0.00056 ± 0.00004
</oasis:entry><oasis:entry colname="5">0.87 ± 0.23
</oasis:entry><oasis:entry colname="6">0.00064
</oasis:entry><oasis:entry colname="7">0.00043
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">AZTR
</oasis:entry><oasis:entry colname="4">0.0028 ± 0.0002
</oasis:entry><oasis:entry colname="5">0.32 ± 0.10
</oasis:entry><oasis:entry colname="6">0.0088
</oasis:entry><oasis:entry colname="7">0.0025
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">DNA−RNA
</oasis:entry><oasis:entry colname="2">PP<sub>i</sub></oasis:entry><oasis:entry colname="3">WT
</oasis:entry><oasis:entry colname="4">0.098 ± 0.006
</oasis:entry><oasis:entry colname="5">1.8 ± 0.3
</oasis:entry><oasis:entry colname="6">0.054
</oasis:entry><oasis:entry colname="7">0.0064
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">AZTR
</oasis:entry><oasis:entry colname="4">0.014 ± 0.001
</oasis:entry><oasis:entry colname="5">0.21 ± 0.04
</oasis:entry><oasis:entry colname="6">0.067
</oasis:entry><oasis:entry colname="7">0.0052
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">ATP
</oasis:entry><oasis:entry colname="3">WT
</oasis:entry><oasis:entry colname="4">0.00017 ± 0.00001
</oasis:entry><oasis:entry colname="5">−<italic toggle="yes"><sup>d</sup></italic><sup></sup></oasis:entry><oasis:entry colname="6">−<italic toggle="yes"><sup>d</sup></italic><sup></sup></oasis:entry><oasis:entry colname="7">0.00015
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">AZTR
</oasis:entry><oasis:entry colname="4">0.0022 ± 0.0003
</oasis:entry><oasis:entry colname="5">1.3 ± 0.5
</oasis:entry><oasis:entry colname="6">0.0017
</oasis:entry><oasis:entry colname="7">0.0015
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">RNA−RNA
</oasis:entry><oasis:entry colname="2">PP<sub>i</sub>/ATP
</oasis:entry><oasis:entry colname="3">WT/AZTR
</oasis:entry><oasis:entry colname="4">no removal observed after 3 h
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> <italic toggle="yes">k</italic><sub>rem</sub> is the maximum rate of nucleotide removal.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Efficiency equals <italic toggle="yes">k</italic><sub>rem</sub>/<italic toggle="yes">K</italic><sub>d</sub> and is expressed in units of s<sup>-1</sup> mM<sup>-1</sup>.<italic toggle="yes"><sup>c</sup></italic><sup></sup> <italic toggle="yes">k</italic><sub>phys</sub> is the calculated rate
of removal at a physiological concentration of the appropriate substrate (125 μM PP<sub>i</sub> or 3 mM ATP).<italic toggle="yes"><sup>d</sup></italic><sup></sup> The kinetic parameter could not be accurately
determined because of insufficient amplitude.</p></table-wrap-foot></table-wrap></p><p>AZT resistant RT mutations can be selected by, and are
cross resistant to, D4T (<italic toggle="yes"><xref rid="bi034435lb00051" ref-type="bibr"></xref></italic>). To understand the kinetics of
D4TMP removal by RT<sup>AZTR</sup>, similar removal studies were
carried out with PP<sub>i</sub> or ATP and a DNA−DNA primer−template (Table <xref rid="bi034435lt00004"></xref>). Only small differences in the kinetic data
were noted between D4TMP and AZTMP removal. Both had
similar efficiencies of removal and predicted physiological
removal rates (<italic toggle="yes">k</italic><sub>phys</sub>), suggesting that, in the absence of other
factors, they are similarly removed by resistant virus. RT<sup>AZTR</sup>
once again had kinetic values similar to those of RT<sup>WT</sup> during
PP<sub>i</sub>lysis but was 1 order of magnitude more efficient during
ATP-mediated removal. For both D4TMP and AZTMP
removal, the <italic toggle="yes">k</italic><sub>phys</sub> (rate constant for removal determined at
physiological concentrations of PP<sub>i</sub> or ATP) values of PP<sub>i</sub>lysis were greater than the rate of ATP-mediated removal
for either RT<sup>WT</sup> or RT<sup>AZTR</sup>.
<table-wrap id="bi034435lt00004" position="float" orientation="portrait"><label>4</label><caption><p>Parameters for D4TMP Removal by ATP- and PP<sub>i</sub>-Mediated Removal</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="7"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:colspec colnum="7" colname="7"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">primer−template</oasis:entry><oasis:entry namest="2" nameend="2">substrate</oasis:entry><oasis:entry namest="3" nameend="3">RT</oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">k</italic><sub>rem</sub> (s<sup>-1</sup>)<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry namest="5" nameend="5"><italic toggle="yes">K</italic><sub>d</sub> (mM)</oasis:entry><oasis:entry namest="6" nameend="6">efficiency<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry namest="7" nameend="7"><italic toggle="yes">k</italic><sub>phys</sub> (s<sup>-1</sup>)<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">DNA−DNA
</oasis:entry><oasis:entry colname="2">PP<sub>i</sub></oasis:entry><oasis:entry colname="3">WT
</oasis:entry><oasis:entry colname="4">0.17 ± 0.02
</oasis:entry><oasis:entry colname="5">1.4 ± 0.4
</oasis:entry><oasis:entry colname="6">0.12
</oasis:entry><oasis:entry colname="7">0.014
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">AZTR
</oasis:entry><oasis:entry colname="4">0.23 ± 0.03
</oasis:entry><oasis:entry colname="5">1.3 ± 0.4
</oasis:entry><oasis:entry colname="6">0.18
</oasis:entry><oasis:entry colname="7">0.020
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">ATP
</oasis:entry><oasis:entry colname="3">WT
</oasis:entry><oasis:entry colname="4">0.00025 ± 0.00001
</oasis:entry><oasis:entry colname="5">0.16 ± 0.07
</oasis:entry><oasis:entry colname="6">0.0016
</oasis:entry><oasis:entry colname="7">0.00024
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">AZTR
</oasis:entry><oasis:entry colname="4">0.0030 ± 0.0003
</oasis:entry><oasis:entry colname="5">0.43 ± 0.15
</oasis:entry><oasis:entry colname="6">0.0070
</oasis:entry><oasis:entry colname="7">0.0026</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> <italic toggle="yes">k</italic><sub>rem</sub> is the maximum rate of nucleotide removal.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Efficiency equals <italic toggle="yes">k</italic><sub>rem</sub>/<italic toggle="yes">K</italic><sub>d</sub> and is expressed in units of s<sup>-1</sup> mM<sup>-1</sup>.<italic toggle="yes"><sup>c</sup></italic><sup></sup> <italic toggle="yes">k</italic><sub>phys</sub> is the rate of removal
at a physiological concentration of the appropriate substrate (125 μM PP<sub>i</sub> or 3 mM ATP).</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">Physiological Role for ATP-Mediated Removal.</italic> (i) The
observation that ATP-mediated removal is catalyzed by RT<sup>WT</sup>
(<italic toggle="yes"><xref rid="bi034435lb00029" ref-type="bibr"></xref></italic>) and (ii) inhibition of blunt-ended addition by PP<sub>i</sub> or ATP
have led to the hypothesis that ATP-mediated removal may
have a physiological role (<italic toggle="yes"><xref rid="bi034435lb00052" ref-type="bibr"></xref></italic>). As a first step in elucidating
this role, experiments were designed to determine the effects
of PP<sub>i</sub> and ATP on several different model systems of
aberrant replication, to provide a quantitative assessment of
the catalytic activities. The effect on either blunt-ended
addition or removal from the 3‘-blunt end or 3‘-overhang
sides of the D21−D22 primer−template was studied in the
presence and absence of PP<sub>i</sub> or ATP. No effect with either
PP<sub>i</sub> or ATP was observed on the slow process of blunt-ended
addition (approximately 10% product formation after 3 h),
and neither PP<sub>i</sub> nor ATP was able to remove the product of
blunt-ended addition (data not shown). Studies with the
D30C−D45R primer−template showed physiological concentrations of PP<sub>i</sub> or ATP to be unable to remove a mismatch.
Direct examination of misincorporation in the presence of
PP<sub>i</sub> or ATP was complicated by the comparable rates of PP<sub>i</sub>lysis and misincorporation. To avoid the shortening of the
primer during the process of misincorporation, a primer−template with a mismatch at the 3‘-end was used (D30C/D45R). RT's misincorporation after a mismatch was found
to be unaffected by physiological concentrations of ATP or
PP<sub>i</sub>. PP<sub>i</sub> was also found to have no effect on correct single-nucleotide incorporation of a natural dNTP at physiological
concentrations (data not shown).
</p><p><italic toggle="yes">Inhibition by the Next Correct Nucleotide on Removal.
</italic>Previous work has shown that the next correct nucleotide
can inhibit ATP-mediated chain terminator removal (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00052" ref-type="bibr"></xref>, <xref rid="bi034435lb00053" ref-type="bibr"></xref></named-content></italic>).
Furthermore, it has been suggested that a decreased level of
next nucleotide inhibition of AZTMP removal relative to
D4TMP removal may account for AZT having a disproportionately greater decrease in activity than D4T when tested
in cell culture with AZT resistant virus (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00052" ref-type="bibr"></xref>−<xref rid="bi034435lb00053" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00054" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00055" ref-type="bibr"></xref></named-content></italic>). To
determine the effect of AZT resistance mutations on the
magnitude and nature of inhibition by the next correct
nucleotide during the pyrophosphorolytic removal of AZTMP
and D4TMP, binding curves for PP<sub>i</sub> were determined in the
presence of varying concentrations of the next correct
nucleotide (Figure <xref rid="bi034435lf00003"></xref>). Differences in competitive and noncompetitive inhibition by the next correct nucleotide were
noted between D4TMP and AZTMP as well as RT<sup>WT</sup> and
RT<sup>AZTR</sup> (Table <xref rid="bi034435lt00005"></xref>). During D4TMP removal, RT<sup>WT</sup> was found
to be competitively inhibited by the next correct nucleotide
while RT<sup>AZTR</sup> showed data consistent with mixed inhibition
(Figure <xref rid="bi034435lf00003"></xref>A). Unlike during D4TMP removal, both RT<sup>WT</sup> and
RT<sup>AZTR</sup> showed noncompetitive inhibition by the next correct
nucleotide during AZTMP removal, and inhibitory concentrations were found to be in great excess over expected
cellular dNTP concentrations (Figure <xref rid="bi034435lf00003"></xref>B). In studies comparing ATP-mediated dNMP removal by RT<sup>AZTR</sup> with D22-D4TMP−D45 and D22-AZTMP−D45 primer−template, it
was observed that at low concentrations of the next correct
nucleotide D4TMP was removed more rapidly than AZTMP
by RT<sup>AZTR</sup> (1.5-fold faster at 1 μM dCTP). Conversely, at
higher next nucleotide concentrations, the rate of AZTMP
removal by RT<sup>AZTR</sup> surpassed that of D4TMP (2.6- and 3.6-fold greater at 25 and 100 μM dCTP, respectively; Figure
<xref rid="bi034435lf00004"></xref>A). These data illustrate the decreased sensitivity of an
AZTMP-terminated primer relative to the D4TMP-terminated
primer with respect to inhibition by the next correct nucleotide during PP<sub>i</sub>- and ATP-mediated removal by RT<sup>WT</sup> and
RT<sup>AZTR</sup>.
<fig id="bi034435lf00003" position="float" orientation="portrait"><label>3</label><caption><p>Inhibition by the next correct nucleotide on AZTMP and D4TMP PP<sub>i</sub>-mediated removal by RT<sup>AZTR</sup>. (A) Determination
of the inhibition constant (<italic toggle="yes">K</italic><sub>i</sub>) for the next correct nucleotide on
D4TMP removal. Hyperbolic curves were generated for the rate
dependence of D4TMP removal on PP<sub>i</sub> concentration in the
presence of 0 (·), 10 (◇), and 50 μM dCTP (▵). The curves are
consistent with mixed inhibition with a noncompetitive inhibition
constant (<italic toggle="yes">K</italic><sub>i</sub><sup>non</sup>) of 39 ± 16 μM and a competitive inhibition constant
(<italic toggle="yes">K</italic><sub>i</sub><sup>com</sup>) of 36 ± 12 μM. (B) Determination of <italic toggle="yes">K</italic><sub>i</sub> for the next correct
nucleotide on AZTMP removal. Hyperbolic curves were generated
for the rate dependence of AZTMP removal on PP<sub>i</sub> concentration
in the presence of 0 (·), 10 (◇), 50 (▵), 100 (×), and 500 μM
dCTP (+). Only noncompetitive inhibition was detected, and <italic toggle="yes">K</italic><sub>i</sub><sup>non</sup>
was equal to 522 ± 244 μM.</p></caption><graphic xlink:href="bi034435lf00003.tif" position="float" orientation="portrait"></graphic></fig><fig id="bi034435lf00004" position="float" orientation="portrait"><label>4</label><caption><p>Inhibition by the next correct nucleotide on AZTMP and D4TMP ATP-mediated (3 mM) removal by RT<sup>WT</sup> and RT<sup>AZTR</sup>.
(A) Comparison of the rates of dNMP removal from the D22-AZTMP−D45 (filled bars) or D22-D4TMP−D45 substrate (empty
bars) by RT<sup>AZTR</sup> at different concentrations of the next correct
nucleotide (dCTP). (B) Percent inhibition of the next correct
nucleotide on ATP-mediated AZTMP removal from the D19-AZTMP−D36 substrate by RT<sup>WT</sup> (◇) and RT<sup>AZTR</sup> (·). The
hyperbolic fits gave IC<sub>50</sub> values of 18 ± 1 and 39 ± 1 μM for
RT<sup>WT</sup> and RT<sup>AZTR</sup>, respectively.</p></caption><graphic xlink:href="bi034435lf00004.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi034435lt00005" position="float" orientation="portrait"><label>5</label><caption><p>Inhibition by the Next Correct Nucleotide (dCTP) on PP<sub>i</sub>- or ATP-Mediated Removal</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="6"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry namest="3" nameend="5">PP<sub>i</sub></oasis:entry><oasis:entry namest="6" nameend="6">ATP</oasis:entry></oasis:row><oasis:row><oasis:entry namest="1" nameend="1">D22-dNMP−D45</oasis:entry><oasis:entry namest="2" nameend="2">RT</oasis:entry><oasis:entry namest="3" nameend="3"><italic toggle="yes">K</italic><sub>i</sub><sup>non</sup>
(μM)<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">K</italic><sub>i</sub><sup>com</sup>
(μM)<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry namest="5" nameend="5">IC<sub>50</sub><sup>phys</sup>
(μM)<italic toggle="yes"><sup>c</sup></italic><sup>,</sup><italic toggle="yes"><sup>d</sup></italic><sup></sup></oasis:entry><oasis:entry namest="6" nameend="6">IC<sub>50</sub><sup>phys</sup>
(μM)<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D4TMP
</oasis:entry><oasis:entry colname="2">WT
</oasis:entry><oasis:entry colname="3">−
</oasis:entry><oasis:entry colname="4">8.2 ± 0.72
</oasis:entry><oasis:entry colname="5">8.8
</oasis:entry><oasis:entry colname="6">7.2 ± 0.8
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AZTR
</oasis:entry><oasis:entry colname="3">39 ± 16
</oasis:entry><oasis:entry colname="4">36 ± 12
</oasis:entry><oasis:entry colname="5">17
</oasis:entry><oasis:entry colname="6">9.9 ± 1.8
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">AZTMP
</oasis:entry><oasis:entry colname="2">WT
</oasis:entry><oasis:entry colname="3">1130 ± 165
</oasis:entry><oasis:entry colname="4">−
</oasis:entry><oasis:entry colname="5">1200
</oasis:entry><oasis:entry colname="6">>500 (18 ± 1<italic toggle="yes"><sup>e</sup></italic><sup></sup>)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AZTR
</oasis:entry><oasis:entry colname="3">522 ± 244
</oasis:entry><oasis:entry colname="4">−
</oasis:entry><oasis:entry colname="5">520
</oasis:entry><oasis:entry colname="6">>500 (39 ± 1<italic toggle="yes"><sup>e</sup></italic><sup></sup>)</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> <italic toggle="yes">K</italic><sub>i</sub> for noncompetitive inhibition.<italic toggle="yes"><sup>b</sup></italic><sup></sup> <italic toggle="yes">K</italic><sub>i</sub> for competitive inhibition.<italic toggle="yes"><sup>c</sup></italic><sup></sup> Concentration necessary to inhibit removal 50% at 125 μM PP<sub>i</sub> or 3 mM
ATP.<italic toggle="yes"><sup>d</sup></italic><sup></sup> Calculated.<italic toggle="yes"><sup>e</sup></italic><sup></sup> Determined with D19-AZTMP−D36 primer−template and dGTP as the next correct nucleotide.</p></table-wrap-foot></table-wrap></p><p>A comparison between RT<sup>WT</sup> and RT<sup>AZTR</sup> showed that
RT<sup>WT</sup> was slightly more sensitive to the next correct
nucleotide than RT<sup>AZTR</sup> during both PP<sub>i</sub>- and ATP-mediated
D4TMP removal (Table <xref rid="bi034435lt00005"></xref>). During AZTMP removal, neither
RT<sup>WT</sup> nor RT<sup>AZTR</sup> was inhibited by the next correct nucleotide
at concentrations of ≤500 μM when using the D22-AZTMP−D45 primer−template. Interestingly, as dCTP
concentrations were increased, an enhancement in the rate
of AZTMP ATP-mediated removal was observed, perhaps
demonstrating dCTP's ability at high concentrations to
catalyze the removal reaction (Figure <xref rid="bi034435lf00004"></xref>A) (<italic toggle="yes"><xref rid="bi034435lb00029" ref-type="bibr"></xref></italic>). Thus, to
assess if there is a difference in the sensitivity to inhibition
by the next correct nucleotide between RT<sup>WT</sup> and RT<sup>AZTR</sup>
for ATP-mediated removal of a chain-terminating AZTMP,
a different sequence context was used. During removal from
an HIV genomically derived D19-AZTMP−D36 primer−template, RT<sup>AZTR</sup> was found to be 2-fold less sensitive to
the next correct nucleotide (dGTP) than RT<sup>WT</sup> (Figure <xref rid="bi034435lf00004"></xref>B).
</p><p><italic toggle="yes">Structure−Activity Relationships Based on Removal of
Cytidine Analogues. </italic>The association of TAMs with multidrug
resistance makes it evident that an understanding of how
nucleotide structural features interact with RT<sup>AZTR</sup> is critical
for the creation of new drugs that are less susceptible to
resistance. A considerable effort has been made to obtain a
detailed structure−activity relationship for the incorporation
of cytidine analogues by both RT<sup>WT</sup> (<italic toggle="yes">35</italic>,<italic toggle="yes"> 56</italic>,<italic toggle="yes"> 57</italic>) and RT<sup>M184V</sup>
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00057" ref-type="bibr"></xref>, <xref rid="bi034435lb00058" ref-type="bibr"></xref></named-content></italic>). Here we studied the rate of removal of three
structurally diverse cytidine analogues by RT<sup>AZTR</sup> and
physiological concentrations of PP<sub>i</sub> or ATP. To avoid error
in comparisons due to the differential establishment of
equilibrium, removal is represented by the initial velocity
derived from a line tangent to the initial phase of the
exponential decay curve. D4CMP was found to have initial
velocities of removal similar to those found with AZTMP
and D4TMP (Table <xref rid="bi034435lt00006"></xref>). All compounds that were studied for
removal showed 7−27-fold faster rates of PP<sub>i</sub>-mediated than
ATP-mediated removal with RT<sup>AZTR</sup>, except 3TC where PP<sub>i</sub>-mediated removal was 80-fold faster than ATP-mediated
removal. The order of effectiveness for removal of cytidine
analogues was found to be as follows: D4CMP > ddCMP
> 3TCMP (Figure <xref rid="bi034435lf00005"></xref>).
<fig id="bi034435lf00005" position="float" orientation="portrait"><label>5</label><caption><p>Structure−activity relationship for the removal of cytidine analogues. Removal studies were carried out with a D23−D45 primer−template where the 3‘-terminal nucleotide was either ddCMP (▾), D4CMP (·), or 3TCMP (○). (A) Removal by RT<sup>AZTR</sup>
in the presence of 125 μM PP<sub>i</sub>. The initial velocities of nucleotide
removal were 0.054, 0.20, and 0.032 nM/s<sup>-1</sup>, respectively. (B)
Removal by RT<sup>AZTR</sup> in the presence of 3 mM ATP. The initial
velocities of nucleotide removal were 0.002, 0.015, and 0.0004 nM/s<sup>-1</sup>, respectively.</p></caption><graphic xlink:href="bi034435lf00005.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi034435lt00006" position="float" orientation="portrait"><label>6</label><caption><p>Initial Velocity for the Removal of Cytidine Analogues from a D23−D45 Primer−Template by RT<sup>AZTR</sup> and PP<sub>i</sub> or ATP</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="3"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry namest="2" nameend="3">initial velocity (nM/s)<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry namest="1" nameend="1">chain terminator</oasis:entry><oasis:entry namest="2" nameend="2">PP<sub>i</sub><italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry namest="3" nameend="3">ATP<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">AZTMP
</oasis:entry><oasis:entry colname="2">0.22
</oasis:entry><oasis:entry colname="3">0.036
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D4TMP
</oasis:entry><oasis:entry colname="2">0.52
</oasis:entry><oasis:entry colname="3">0.039
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">ddCMP
</oasis:entry><oasis:entry colname="2">0.054
</oasis:entry><oasis:entry colname="3">0.002
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D4CMP
</oasis:entry><oasis:entry colname="2">0.20
</oasis:entry><oasis:entry colname="3">0.015
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">3TCMP
</oasis:entry><oasis:entry colname="2">0.032
</oasis:entry><oasis:entry colname="3">0.0004</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> The velocity before the rapid attainment of equilibrium.<italic toggle="yes"><sup>b</sup></italic><sup></sup> PP<sub>i</sub>lysis
reactions were carried out in the presence of 125 μM PP<sub>i</sub> and ATP
removal reactions in the presence of 3 mM ATP, except for ATP
removal of AZTMP and D4TMP which were carried out at 2.5 and 2
mM ATP, respectively.</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">Efficiency of TMP and AZTMP Incorporation by RT<sup>WT</sup></italic><sup></sup><italic toggle="yes"> and
RT<sup>AZTR</sup></italic><sup></sup><italic toggle="yes"> during the Initiation of HIV Reverse Transcription.</italic>
Slight changes in the incorporation efficiency for RT<sup>WT</sup> and
RT<sup>AZTR</sup> during RNA-directed processes have been observed
in previous work by our laboratory (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00027" ref-type="bibr"></xref>, <xref rid="bi034435lb00028" ref-type="bibr"></xref></named-content></italic>). Incorporation
studies with a model 18-mer RNA primer representing the
priming sequence from tRNA<sub>3</sub><sup>Lys</sup> and a 36-mer RNA template
containing the primer binding site found that low-level
selectivity against AZTMP incorporation was conferred by
AZT resistance mutations. This finding led to the hypothesis
that increased selectivity on the part of HIV-1 RT<sup>AZTR</sup> during
initiation may play a part in AZT resistance (<italic toggle="yes"><xref rid="bi034435lb00027" ref-type="bibr"></xref></italic>). To further
test this hypothesis, pre-steady-state single-turnover experiments were used to compare the incorporation of TMP with
AZTMP by RT<sup>WT</sup> and RT<sup>AZTR</sup> into a model initiation system
that included a synthetic tRNA<sub>3</sub><sup>Lys</sup> primer and an R36
template. The observed rates from single-turnover experiments were plotted against dNTP concentration to determine
the maximum rate of incorporation (<italic toggle="yes">k</italic><sub>pol</sub>) and binding constant
for productive complex formation (<italic toggle="yes">K</italic><sub>d</sub>), and these values were
used to calculate the efficiency of incorporation in the
presence of either TTP or AZTTP (Table <xref rid="bi034435lt00007"></xref> and Figure <xref rid="bi034435lf00006"></xref>).
Only slight differences were recognized between RT<sup>WT</sup> and
RT<sup>AZTR</sup>, but consistent with previous results, AZTMP was
less efficiently incorporated by RT<sup>AZTR</sup> than by RT<sup>WT</sup>. These
experiments revealed data that were very similar to those
found with the R18-mer primer used previously (<italic toggle="yes"><xref rid="bi034435lb00027" ref-type="bibr"></xref></italic>),
suggesting that the rest of the tRNA sequence does not
enhance selectivity between TTP and AZTTP in the presence
of synthetic tRNA<sub>3</sub><sup>Lys</sup>.
<fig id="bi034435lf00006" position="float" orientation="portrait"><label>6</label><caption><p>Dependence of the first-order rate constant (<italic toggle="yes">k</italic><sub>obsd</sub>) for
incorporation into a synthetic tRNA<sub>3</sub><sup>Lys</sup>−R36 primer−template on
TTP (A) or AZTTP (B) concentration for RT<sup>WT</sup> (·) and RT<sup>AZTR</sup>
(○). The observed rates of dNMP incorporation into the model
initiation complex were plotted against the concentration of dNTP
and fit to hyperbolic equations. The fit showed that TMP was
incorporated with maximum rates (<italic toggle="yes">k</italic><sub>pol</sub>) of 0.46 ± 0.02 and 0.64 ±
0.06 s<sup>-1</sup> and binding constants (<italic toggle="yes">K</italic><sub>d</sub>) of 84 ± 13 and 130 ± 30 μM
for RT<sup>WT</sup> and RT<sup>AZTR</sup>, respectively. AZTMP had <italic toggle="yes">k</italic><sub>pol</sub> values of 0.27
± 0.01 and 0.24 ± 0.01 s<sup>-1</sup> and <italic toggle="yes">K</italic><sub>d</sub> values of 170 ± 20 and 240 ±
20 μM for RT<sup>WT</sup> and RT<sup>AZTR</sup>, respectively.</p></caption><graphic xlink:href="bi034435lf00006.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi034435lt00007" position="float" orientation="portrait"><label>7</label><caption><p>Kinetic Constants for Binding and Incorporation of TTP or AZTTP with a Synthetic tRNA<sub>3</sub><sup>Lys</sup>−R36 Primer−Template and
RT<sup>WT</sup> or RT<sup>AZTR</sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="6"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">RT</oasis:entry><oasis:entry namest="2" nameend="2">nucleotide</oasis:entry><oasis:entry namest="3" nameend="3"><italic toggle="yes">k</italic><sub>pol</sub> (s<sup>-1</sup>)</oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">K</italic><sub>d</sub> (μM)</oasis:entry><oasis:entry namest="5" nameend="5">efficiency<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry namest="6" nameend="6">selectivity<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">WT
</oasis:entry><oasis:entry colname="2">TTP
</oasis:entry><oasis:entry colname="3">0.46 ± 0.02
</oasis:entry><oasis:entry colname="4">84 ± 13
</oasis:entry><oasis:entry colname="5">0.0055 ± 0.0009
</oasis:entry><oasis:entry colname="6">3.4 ± 0.7
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AZTTP
</oasis:entry><oasis:entry colname="3">0.27 ± 0.01
</oasis:entry><oasis:entry colname="4">170 ± 20
</oasis:entry><oasis:entry colname="5">0.0015 ± 0.0002
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">AZTR
</oasis:entry><oasis:entry colname="2">TTP
</oasis:entry><oasis:entry colname="3">0.64 ± 0.06
</oasis:entry><oasis:entry colname="4">130 ± 30
</oasis:entry><oasis:entry colname="5">0.0049 ± 0.0013
</oasis:entry><oasis:entry colname="6">4.9 ± 1.4
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AZTTP
</oasis:entry><oasis:entry colname="3">0.24 ± 0.01
</oasis:entry><oasis:entry colname="4">240 ± 20
</oasis:entry><oasis:entry colname="5">0.0010 ± 0.0001
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Efficiency equals <italic toggle="yes">k</italic><sub>pol</sub>/<italic toggle="yes">K</italic><sub>d</sub> and is expressed in s<sup>-1</sup> μM<sup>-1</sup>.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Selectivity
= efficiency<sup>TTP</sup>/efficiency<sup>AZTTP</sup>.</p></table-wrap-foot></table-wrap></p></sec><sec id="d7e2550"><title>Discussion</title><p><italic toggle="yes">Mechanism of Removal of Nucleotides by RT<sup>WT</sup></italic><sup></sup><italic toggle="yes"> and
RT<sup>AZTR</sup></italic><sup></sup><italic toggle="yes">. </italic>The comparison of PP<sub>i</sub>lysis and PP<sub>i</sub> exchange for
RT<sup>WT</sup> and RT<sup>AZTR</sup> suggests that HIV-1 RT, like other DNA
polymerases, has a rate-limiting step for the reverse reaction
that occurs after PP<sub>i</sub> binding. The lack of a difference
between RT<sup>WT</sup> and RT<sup>AZTR</sup> in PP<sub>i</sub>lysis and PP<sub>i</sub> exchange
suggests that TAMs do not have any marked effect on the
rate or the mechanism of the reverse reaction when PP<sub>i</sub> is
responsible for the removal of the terminal dNMP. Studies
were also carried out to further identify the rate-limiting step
of the reaction through experiments using a nucleotide in
which one of the α-phosphate oxygens is substituted with a
sulfur. A much larger phosphorothioate effect was noted
during removal in comparison to incorporation [where it is
believed that a rate-limiting step precedes chemistry (<italic toggle="yes"><xref rid="bi034435lb00036" ref-type="bibr"></xref></italic>)],
suggesting that chemistry is rate-limiting during removal.
However, the elemental effect has been illustrated as an
unreliable indicator of rate-limiting chemistry by the measurement of a smaller elemental effect for a reaction closely
related to polymerization (<italic toggle="yes"><xref rid="bi034435lb00050" ref-type="bibr"></xref></italic>). Issues surrounding the
elemental effect have been discussed at length in other papers
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00059" ref-type="bibr"></xref>, <xref rid="bi034435lb00060" ref-type="bibr"></xref></named-content></italic>). In light of these arguments, it can only be suggested
that the rate-limiting step in the reverse reaction is the
chemical step, and in Scheme <xref rid="bi034435lh00001"></xref> (model 1), the rate-limiting
step (<italic toggle="yes">k</italic><sub>pyro</sub>) is loosely defined as either the chemical step or
a preceding conformation change which includes the RT·primer−template·PP<sub>i</sub> complex. From the slow relative rate
of removal in comparison to the rate of primer−template
release, it can also be inferred that the rate of primer−template release and reassociation may also play a critical
role in dictating the maximum rate of removal. These
parameters have been found to be affected by TAMs and a
chain-terminating AZTMP (<italic toggle="yes"><xref rid="bi034435lb00061" ref-type="bibr"></xref></italic>), and changes in the dissociation rate of chain-terminated primer−templates from RT have
been suggested as a mechanism for enhancing removal by
mutants resistant to abacavir (<italic toggle="yes"><xref rid="bi034435lb00039" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Comparison of Removal Kinetics for AZTMP and D4TMP
by RT<sup>WT</sup></italic><sup></sup><italic toggle="yes"> and RT<sup>AZTR</sup></italic><sup></sup> <italic toggle="yes">Using either PP<sub>i</sub></italic><italic toggle="yes">- or ATP-Mediated
Removal during Different Stages of Viral Replication.</italic> Kinetic
constants for the removal of AZTMP and D4TMP were
found to be very similar. For both analogues, the maximum
rate of PP<sub>i</sub>lysis (<italic toggle="yes">k</italic><sub>rem</sub>) and the physiologically expected rate
of removal (<italic toggle="yes">k</italic><sub>phys</sub>) were always faster than the corresponding
rates of ATP-mediated removal, independent of the protein
that was used. However, the rate of PP<sub>i</sub>lysis was not increased
by TAMs, while the rate of ATP-dependent removal was
enhanced as much as 10-fold. On the basis of the observed
biochemical differences of the removal reaction between
RT<sup>WT</sup> and RT<sup>AZTR</sup>, it may be suggested that ATP is the
relevant mediator of resistance. It was notable, however, that
with RT<sup>WT</sup> under estimated physiological concentrations of
PP<sub>i</sub> (125 μM) or ATP (3 mM), PP<sub>i</sub>lysis occurs at a rate 40-
to 60-fold faster than the rate of ATP-mediated removal for
AZTMP and D4TMP removal (calculated from values
presented in Tables <xref rid="bi034435lt00003"></xref> and <xref rid="bi034435lt00004"></xref>). This difference was only
reduced for RT<sup>AZTR</sup> where PP<sub>i</sub>lysis was on average 5-fold
faster than ATP-mediated removal of AZTMP and D4TMP
(taking into account the values for cytidine analogues
presented in Table <xref rid="bi034435lt00006"></xref>, we determine the average is 20-fold).
Results showing that the AZT resistant virus is more sensitive
to foscarnet (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00023" ref-type="bibr"></xref>, <xref rid="bi034435lb00062" ref-type="bibr"></xref></named-content></italic>), a PP<sub>i</sub> analogue, would also be
seemingly unexplained without the involvement of PP<sub>i</sub> in
the resistance mechanism. In light of the faster rate of PP<sub>i</sub>lysis and the enhanced sensitivity of the resistant virus to
foscarnet, it is reasonable to believe that both removal
substrates are responsible for resistance and perhaps other
biochemical properties mediate PP<sub>i</sub>'s role in AZT resistance.
</p><p>Most of the research that has been done to date has focused
on the effects of TAMs on removal of nucleotides from a
DNA−DNA primer−template. However, removal during
RNA-directed incorporation would be critical in resistance
caused by TAMs. Comparison of the efficiencies of removal
of AZTMP from a DNA−DNA, DNA−RNA, and RNA−RNA primer−template shows that removal is less efficient
during RNA-directed processes, and no analogue removal
was observed from our model RNA19-AZTMP−R36 initiation system. These results are consistent with the lack of
removal observed with a tRNA<sub>3</sub><sup>Lys</sup> chain-terminated primer
by others (<italic toggle="yes"><xref rid="bi034435lb00063" ref-type="bibr"></xref></italic>) and illustrates that AZT resistant virus may
be more sensitive to chain termination during the RNA-directed process, especially initiation.
</p><p><italic toggle="yes">A Physiological Role for ATP-Dependent Removal during
Normal Viral Replication?</italic> It is unclear whether ATP may
influence RT fidelity either by decreasing the extent of
aberrant incorporation or removing its products. It has been
suggested that ATP-dependent removal may play a role in
limiting blunt-ended addition (<italic toggle="yes"><xref rid="bi034435lb00052" ref-type="bibr"></xref></italic>). The slow rate of blunt-ended addition, however, raises doubt about its physiological
relevance. Results presented in this paper also show ATP to
be unable to directly affect blunt-ended addition or remove
a 3‘-overhang. An alternative explanation for the results
observed previously is that a decreased level of blunt-ended
addition was due to an inhibition of processive synthesis
caused by PP<sub>i</sub> or high concentrations of ATP. This would
also be consistent with an overall inhibition of multiple
incorporations observed by others in the presence of PP<sub>i</sub> or
ATP (<italic toggle="yes"><xref rid="bi034435lb00064" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">TAMs Play a Critical Role in the Optimal Alignment of a
Chain-Terminated Primer−Template and ATP Rather than
Increasing the Extent of ATP Binding.</italic> The kinetic data
summarized in Table <xref rid="bi034435lt00003"></xref> illustrate that TAMs cause the greatest
effect on the rate of removal, suggesting that ATP is more
effectively positioned for catalytic attack of the terminating
dNMP. An early report suggested that the mutations at
positions 67 and 70 are important in dictating the enhanced
rate of ATP-mediated removal (<italic toggle="yes"><xref rid="bi034435lb00053" ref-type="bibr"></xref></italic>), and perhaps these
residues are responsible for positioning ATP and the chain-terminated primer−template for effective removal. Our data
are not consistent with the hypothesis that ATP is bound
more tightly by RT<sup>AZTR</sup> (<italic toggle="yes"><xref rid="bi034435lb00052" ref-type="bibr"></xref></italic>). During DNA−DNA removal
of AZTMP, RT<sup>AZTR</sup> only bound 2.7-fold tighter to ATP than
RT<sup>WT</sup> (Table <xref rid="bi034435lt00003"></xref>), while during D4TMP removal, 2.7-fold
weaker binding was observed (Table <xref rid="bi034435lt00004"></xref>), showing no consistent trend.
</p><p><italic toggle="yes">Effects of TAMs on the Inhibition of Removal by the Next
Correct Nucleotide.</italic> It has been shown that the next correct
incoming nucleotide can inhibit chain terminator removal
most likely by causing RT to translocate to a position one
nucleotide past the chain terminator, no longer allowing for
cleavage of the newly formed phosphodiester bond (<italic toggle="yes"><xref rid="bi034435lb00053" ref-type="bibr"></xref></italic>).
Consistent with previous data (<italic toggle="yes"><xref rid="bi034435lb00054" ref-type="bibr"></xref></italic>), AZTMP was found to
be 50-fold less sensitive to inhibition by the next correct
nucleotide than D4TMP during both pyrophosphorolysis and
ATP-mediated removal by either RT<sup>WT</sup> or RT<sup>AZTR</sup> (Table <xref rid="bi034435lt00005"></xref>
and Figure <xref rid="bi034435lf00004"></xref>A). These results illustrate RT's sensitivity to
nucleotide analogue structural features during removal
(discussed further below).
</p><p>Decreased sensitivity to the next correct nucleotide may
also serve as a mechanism for increasing resistance of RT
mutants by allowing them to more efficiently remove chain
terminators in a cellular environment where the next deoxynucleotide is present. Residues 215 and 219 have been
implicated in altering the interaction with the incoming
nucleotide through biochemical (<italic toggle="yes"><xref rid="bi034435lb00053" ref-type="bibr"></xref></italic>), molecular modeling
(<italic toggle="yes"><xref rid="bi034435lb00065" ref-type="bibr"></xref></italic>), and crystallographic (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00066" ref-type="bibr"></xref>, <xref rid="bi034435lb00067" ref-type="bibr"></xref></named-content></italic>) findings. Data presented
here show changes in the interaction of RT<sup>AZTR</sup> with the chain
terminator as well as the incoming next correct nucleotide
during removal. The decreased level of inhibition of the next
correct nucleotide on PP<sub>i</sub>-mediated removal of D4TMP by
RT<sup>AZTR</sup> may illustrate how, in the absence of large changes
in rate or binding of PP<sub>i</sub>, PP<sub>i</sub>lysis may play a role in
resistance. Although sequence-dependent differences were
apparent, decreased sensitivity due to mutations present in
RT<sup>AZTR</sup> during ATP-mediated removal of D4TMP and
AZTMP were also obtained. These data are consistent with
results showing a decreased level of inhibition by the next
correct nucleotide during ATP-mediated removal of AZTMP
and ddAMP in response to TAMs by others (<italic toggle="yes"><xref rid="bi034435lb00053" ref-type="bibr"></xref></italic>). Taken
together, these results suggest that a decreased level of
inhibition by the next correct nucleotide may allow mutant
RT to utilize the more rapid process of PP<sub>i</sub>-mediated removal
to enhance resistance as well as minimize inhibition of ATP-mediated removal in a cellular environment.
</p><p><italic toggle="yes">Structure−Activity Relationships for the Removal of
Cytidine Analogues.</italic> The removal of structurally distinct
cytidine analogues showed that like D4TMP chain-terminated
primers, D4CMP chain-terminated primers are also prone
to efficient removal by RT<sup>AZTR</sup> with either PP<sub>i</sub> or ATP. From
these results, a structure−activity relationship begins to
emerge for ribose ring modifications which are more prone
to RT<sup>AZTR</sup> removal. Kinetic assays have shown the planar
unsaturated analogues D4TMP, D4CMP, and CBVMP are
subject to efficient removal. In a recent report, we have
hypothesized that the correlation between abacavir resistance
and TAMs may be due to CBVMP forming strong hydrophobic interactions in the active site, causing it to remain in
a position competent for removal (<italic toggle="yes"><xref rid="bi034435lb00039" ref-type="bibr"></xref></italic>). Both D4TMP and
D4CMP may also form strong π−π stacking interactions
with Tyr115 and be more likely to occupy the RT active
site in a favorable position for removal to take place.
AZTMP, however, does not have the same potential for
hydrophobic interactions within the active site, but its large
azido group may allow for many electrostatic and hydrogen
bonding contacts within the 3‘-hydroxyl pocket of the active
site. These could act to improve positioning for the removal
reaction as well as anchor AZTMP in the active site,
disfavoring translocation and inhibition by the next correct
nucleotide. The potential for these interactions is evident in
the recently reported crystal structure of RT with AZTMP
in the nucleotide binding site (<italic toggle="yes"><xref rid="bi034435lb00068" ref-type="bibr"></xref></italic>). It has also been
hypothesized that the azido group sterically hinders the next
incoming nucleotide (<italic toggle="yes"><xref rid="bi034435lb00052" ref-type="bibr"></xref></italic>) and this and/or tight binding may
cause AZTMP chain-terminated primers to be superior
substrates for removal and less sensitive to inhibition by the
next incoming dNTP.
</p><p><italic toggle="yes">Increased Selectivity by RT<sup>AZTR</sup></italic><sup></sup><italic toggle="yes"> during Incorporation into
a Model Initiation System. </italic>Similar to previous studies on
RNA-dependent incorporation, a slight increase in selectivity
was seen for RT<sup>AZTR</sup> in our model initiation system (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00027" ref-type="bibr"></xref>, <xref rid="bi034435lb00028" ref-type="bibr"></xref></named-content></italic>).
The similarities between the kinetic constants determined
with the full tRNA<sub>3</sub><sup>Lys</sup> sequence and those determined with
an 18-mer RNA (<italic toggle="yes"><xref rid="bi034435lb00027" ref-type="bibr"></xref></italic>) suggest that interactions with the
additional nucleotide sequence do not play a role in
resistance. It is possible that other factors present in the
initiation complex, including modified nucleotides present
in natural tRNA<sub>3</sub><sup>lys</sup>, the virally encoded nucleocapsid protein
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi034435lb00069" ref-type="bibr"></xref>−<xref rid="bi034435lb00070" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi034435lb00071" ref-type="bibr"></xref></named-content></italic>), or the phosphorylated state of RT (<italic toggle="yes"><xref rid="bi034435lb00072" ref-type="bibr"></xref></italic>), may
modulate AZT resistance. Further in-depth studies may yield
interesting information about the initiation process and its
potential role in NRTI resistance. Data showing the inefficient removal of chain terminators during RNA-directed
processes illustrate the potential importance of even slight
selectivity advantages in allowing resistant virus to replicate
in the presence of nucleotide analogues.
</p></sec><sec id="d7e2876"><title>Conclusions</title><p>Our studies have shown that AZT resistance mutations
cause a more effective positioning of substrates for the
catalysis of ATP-mediated removal. It was also found that a
decreased level of interactions with the next correct nucleotide during D4TMP removal by RT<sup>AZTR</sup> may illustrate how
PP<sub>i</sub> can play a role in resistance despite no marked increase
in the pyrophosphorylitic rate of removal. Decreased sensitivity to the next correct nucleotide was also shown to serve
as a potential mechanism for an increased level of ATP-mediated removal. Because of RT's weakened ability to
remove nucleotide analogues during RNA-directed processes,
the slight differences in selectivity noted during AZTMP
incorporation might be an important factor in enhancing
resistance. Indeed, our data illustrate that increased selectivity, PP<sub>i</sub>lysis, and ATP-mediated removal may all contribute
to the overall magnitude of resistance observed in a cellular
system. An initial structure−activity relationship suggests
that nucleotide analogues with the potential to form strong
interactions with the RT active site may be more prone to
resistance both by being better substrates for the removal
reaction and perhaps by having their removal less inhibited
by the next correct incoming nucleotide. A deeper understanding of the effects of nucleotide features on removal may
allow for the rational design of new and more effective agents
against resistant HIV.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank Stephen Hughes, Paul Boyer, and Andrea Ferris
for the HIV-1 RT<sup>WT</sup> and RT<sup>AZTR</sup> clones and Thomas A. Steitz
and Janis Pata for the synthetic tRNA expression system.
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The suffixes -MP and -TP are added to the drug abbreviations to indicate their monophosphate and triphosphate forms, respectively.</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1 Reverse Transcriptase Using a Transient Kinetic Analysis†</title></titleInfo><titleInfo contentType="CDATA"><title>Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1 Reverse Transcriptase Using a Transient Kinetic Analysis†</title></titleInfo><name type="personal"><namePart type="family">RAY</namePart><namePart type="given">Adrian S.</namePart><affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</affiliation><affiliation> Yale University School of Medicine.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">MURAKAMI</namePart><namePart type="given">Eisuke</namePart><affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</affiliation><affiliation> Yale University School of Medicine.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">BASAVAPATHRUNI</namePart><namePart type="given">Aravind</namePart><affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</affiliation><affiliation> Yale University School of Medicine.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">VACCARO</namePart><namePart type="given">Joseph A.</namePart><affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</affiliation><affiliation> Yale University School of Medicine.</affiliation><affiliation> Current address: Department of Biochemistry, Tulane UniversityHealth Sciences Center, 1430 Tulane Ave. SL-43, New Orleans, LA70112.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">ULRICH</namePart><namePart type="given">Dagny</namePart><affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</affiliation><affiliation> Yale University School of Medicine.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">CHU</namePart><namePart type="given">Chung K.</namePart><affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</affiliation><affiliation> The University of Georgia.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">SCHINAZI</namePart><namePart type="given">Raymond F.</namePart><affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</affiliation><affiliation> Emory University School of Medicine/Veterans Affairs MedicalCenter.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">ANDERSON</namePart><namePart type="given">Karen S.</namePart><affiliation>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033</affiliation><affiliation> Yale University School of Medicine.</affiliation><affiliation> To whom correspondence should be addressed. E-mail: karen.anderson@yale.edu. Phone: (203) 785-4526. Fax: (203) 785-7670.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">2003-07-04</dateCreated><dateIssued encoding="w3cdtf">2003-07-29</dateIssued><copyrightDate encoding="w3cdtf">2003</copyrightDate></originInfo><note type="footnote" ID="bi034435lAF2"> Work supported by NIH Grants GM49551 (K.S.A.), R37AI-41980 (R.F.S.), AI 25899 (C.K.C.), and RO1AI-32351 (R.F.S. and C.K.C.), the Department of Veteran Affairs (R.F.S.), NRSA 5 T32 GM07223 from the National Institute of General Medical Sciences (A.S.R.), and ACS Postdoctoral Fellowship PF-4478 (J.A.V.).</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase (RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision, suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain terminator from an RNA−RNA primer−template may show how slight changes in selectivity against AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance. Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations. Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.</abstract><note type="footnote" ID="bi034435lAF2"> Work supported by NIH Grants GM49551 (K.S.A.), R37AI-41980 (R.F.S.), AI 25899 (C.K.C.), and RO1AI-32351 (R.F.S. and C.K.C.), the Department of Veteran Affairs (R.F.S.), NRSA 5 T32 GM07223 from the National Institute of General Medical Sciences (A.S.R.), and ACS Postdoctoral Fellowship PF-4478 (J.A.V.).</note><relatedItem type="host"><titleInfo><title>Biochemistry</title></titleInfo><titleInfo type="abbreviated"><title>Biochemistry</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">0006-2960</identifier><identifier type="eISSN">1520-4995</identifier><identifier type="acspubs">bi</identifier><identifier type="coden">BICHAW</identifier><identifier type="uri">pubs.acs.org/biochemistry</identifier><part><date>2003</date><detail type="volume"><caption>vol.</caption><number>42</number></detail><detail type="issue"><caption>no.</caption><number>29</number></detail><extent unit="pages"><start>8831</start><end>8841</end></extent></part></relatedItem><relatedItem type="references" ID="bi034435lb00001" displayLabel="bibbi034435lb00001"><name type="personal"><namePart type="family">TRAUT</namePart><namePart type="given">T. 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Commun.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Lazaro J. B. Boretto J. Selmi B. Capony J. P. Canard B. Biochem. Biophys. Res. Commun. 2000 275 26 32 10.1006/bbrc.2000.3251</note><identifier type="doi">10.1006/bbrc.2000.3251</identifier><part><date>2000</date><detail type="volume"><caption>vol.</caption><number>275</number></detail><extent unit="pages"><start>26</start><end>32</end></extent></part></relatedItem><relatedItem type="references" ID="bi034435ln00001" displayLabel="bibbi034435ln00001"><titleInfo><title>Abbreviations: RT, reverse transcriptase; NRTI, nucleoside reverse transcriptase inhibitor; dNMP, 2‘-deoxynucleoside 5‘-monophosphate; dNTP, 2‘-deoxynucleoside 5‘-triphosphate; abacavir, (1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol succinate; AZT, β-d-(+)-3‘-azido-3‘-deoxythymidine; CBV, carbovir; D4T, β-d-(+)-2‘,3‘-didehydro-3‘-deoxythymidine; 3TC, β-l-(−)-2‘,3‘-dideoxy-3‘-thiacytidine; ddC, β-d-(+)-2‘,3‘-dideoxycytidine; D4C, β-d-(+)-2‘,3‘-didehydro-2‘,3‘-dideoxycytidine; PMPA, (R)-9-(2-phosphonylmethoxypropyl)adenine; PPi, pyrophosphate; PPilysis, pyrophosphorolysis; phys, values that represent kinetic constants at physiological concentrations of PPi(125 μM) or ATP (3 mM, concentrations from ref1); WT, wild type; TAMs, thymidine-associated mutations; AZTR, AZT resistant quadruple mutant containing D67N, K70R, T215Y, and K219Q mutations. The suffixes -MP and -TP are added to the drug abbreviations to indicate their monophosphate and triphosphate forms, respectively.</title></titleInfo><note type="content-in-line">Abbreviations: RT, reverse transcriptase; NRTI, nucleoside reverse transcriptase inhibitor; dNMP, 2‘-deoxynucleoside 5‘-monophosphate; dNTP, 2‘-deoxynucleoside 5‘-triphosphate; abacavir, (1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol succinate; AZT, β-d-(+)-3‘-azido-3‘-deoxythymidine; CBV, carbovir; D4T, β-d-(+)-2‘,3‘-didehydro-3‘-deoxythymidine; 3TC, β-l-(−)-2‘,3‘-dideoxy-3‘-thiacytidine; ddC, β-d-(+)-2‘,3‘-dideoxycytidine; D4C, β-d-(+)-2‘,3‘-didehydro-2‘,3‘-dideoxycytidine; PMPA, (R)-9-(2-phosphonylmethoxypropyl)adenine; PPi, pyrophosphate; PPilysis, pyrophosphorolysis; phys, values that represent kinetic constants at physiological concentrations of PPi (125 μM) or ATP (3 mM, concentrations from ref 1); WT, wild type; TAMs, thymidine-associated mutations; AZTR, AZT resistant quadruple mutant containing D67N, K70R, T215Y, and K219Q mutations. The suffixes -MP and -TP are added to the drug abbreviations to indicate their monophosphate and triphosphate forms, respectively.</note></relatedItem><identifier type="istex">27E0B499B0C8B79DCFA4C54E465DD77F7037A1FA</identifier><identifier type="ark">ark:/67375/TPS-1F3PKRB5-X</identifier><identifier type="DOI">10.1021/bi034435l</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2003 American Chemical Society</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">acs</recordContentSource><recordOrigin>Converted from (version 1.2.10) to MODS version 3.6.</recordOrigin><recordCreationDate encoding="w3cdtf">2020-04-10</recordCreationDate></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-1F3PKRB5-X/record.json</uri></json:item></metadata><annexes><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/27E0B499B0C8B79DCFA4C54E465DD77F7037A1FA/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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