Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities
Identifieur interne : 001A54 ( Istex/Corpus ); précédent : 001A53; suivant : 001A55Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities
Auteurs : Cathryn A. Shaw-Reid ; Bradley Feuston ; Vandna Munshi ; Krista Getty ; Julie Krueger ; Daria J. Hazuda ; Michael A. Parniak ; Michael D. Miller ; Dale LewisSource :
- Biochemistry [ 0006-2960 ] ; 2005.
Abstract
Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3‘-azido-3‘-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.
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DOI: 10.1021/bi0486740
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Combinations on HIV-1 Reverse-Transcriptase Activities</title><author><name sortKey="Shaw Reid, Cathryn A" sort="Shaw Reid, Cathryn A" uniqKey="Shaw Reid C" first="Cathryn A." last="Shaw-Reid">Cathryn A. Shaw-Reid</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation></affiliation><affiliation><mods:affiliation> Present address: Amgen, Inc., One Amgen Center Drive, ThousandOaks, CA 91320-1799.</mods:affiliation></affiliation></author><author><name sortKey="Feuston, Bradley" sort="Feuston, Bradley" uniqKey="Feuston B" first="Bradley" last="Feuston">Bradley Feuston</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Molecular Systems, Merck Research Laboratories.</mods:affiliation></affiliation></author><author><name sortKey="Munshi, Vandna" sort="Munshi, Vandna" uniqKey="Munshi V" first="Vandna" last="Munshi">Vandna Munshi</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation></affiliation></author><author><name sortKey="Getty, Krista" sort="Getty, Krista" uniqKey="Getty K" first="Krista" last="Getty">Krista Getty</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation></affiliation></author><author><name sortKey="Krueger, Julie" sort="Krueger, Julie" uniqKey="Krueger J" first="Julie" last="Krueger">Julie Krueger</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Cardiovascular Diseases, Merck Research Laboratories.</mods:affiliation></affiliation></author><author><name sortKey="Hazuda, Daria J" sort="Hazuda, Daria J" uniqKey="Hazuda D" first="Daria J." last="Hazuda">Daria J. Hazuda</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation></affiliation></author><author><name sortKey="Parniak, Michael A" sort="Parniak, Michael A" uniqKey="Parniak M" first="Michael A." last="Parniak">Michael A. Parniak</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Medicine/Division of Infectious Diseases, Universityof Pittsburgh.</mods:affiliation></affiliation></author><author><name sortKey="Miller, Michael D" sort="Miller, Michael D" uniqKey="Miller M" first="Michael D." last="Miller">Michael D. Miller</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed: Department ofAntiviral Research, Merck and Co., Inc., P.O. Box 4, WP42-209, WestPoint, PA 19486-0004. Telephone: 215-652-0480. Fax: 215-993-5798.E-mail: michael_miller1@merck.com.</mods:affiliation></affiliation></author><author><name sortKey="Lewis, Dale" sort="Lewis, Dale" uniqKey="Lewis D" first="Dale" last="Lewis">Dale Lewis</name><affiliation><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Cardiovascular Diseases, Merck Research Laboratories.</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">2005</date><date type="published">2005</date><biblScope unit="vol">44</biblScope><biblScope unit="issue">5</biblScope><biblScope unit="page" from="1595">1595</biblScope><biblScope unit="page" to="1606">1606</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">Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3‘-azido-3‘-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>rnase</json:string><json:string>nnrti</json:string><json:string>polymerase</json:string><json:string>assay</json:string><json:string>transcriptase</json:string><json:string>inhibitor</json:string><json:string>cleavage</json:string><json:string>titrated</json:string><json:string>polymerase inhibitor</json:string><json:string>biochemistry</json:string><json:string>mutation</json:string><json:string>immunodeficiency</json:string><json:string>mutant</json:string><json:string>nnrti binding</json:string><json:string>synergy</json:string><json:string>datum</json:string><json:string>active 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Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</json:string><json:string>Department of Molecular Systems, Merck Research Laboratories.</json:string></affiliations></json:item><json:item><name>MUNSHI Vandna</name><affiliations><json:string>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</json:string><json:string>Department of Antiviral Research, Merck Research Laboratories.</json:string></affiliations></json:item><json:item><name>GETTY Krista</name><affiliations><json:string>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</json:string><json:string>Department of Antiviral Research, Merck Research Laboratories.</json:string></affiliations></json:item><json:item><name>KRUEGER Julie</name><affiliations><json:string>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</json:string><json:string>Department of Cardiovascular Diseases, Merck Research Laboratories.</json:string></affiliations></json:item><json:item><name>HAZUDA Daria J.</name><affiliations><json:string>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</json:string><json:string>Department of Antiviral Research, Merck Research Laboratories.</json:string></affiliations></json:item><json:item><name>PARNIAK Michael A.</name><affiliations><json:string>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</json:string><json:string>Department of Medicine/Division of Infectious Diseases, Universityof Pittsburgh.</json:string></affiliations></json:item><json:item><name>MILLER Michael D.</name><affiliations><json:string>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</json:string><json:string>Department of Antiviral Research, Merck Research Laboratories.</json:string><json:string>To whom correspondence should be addressed: Department ofAntiviral Research, Merck and Co., Inc., P.O. Box 4, WP42-209, WestPoint, PA 19486-0004. Telephone: 215-652-0480. Fax: 215-993-5798.E-mail: michael_miller1@merck.com.</json:string></affiliations></json:item><json:item><name>LEWIS Dale</name><affiliations><json:string>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</json:string><json:string>Department of Cardiovascular Diseases, Merck Research Laboratories.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-6GP69DPW-0</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3‘-azido-3‘-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.</abstract><qualityIndicators><score>9.796</score><pdfWordCount>7820</pdfWordCount><pdfCharCount>48852</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>12</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>652</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>false</refBibsNative><abstractWordCount>233</abstractWordCount><abstractCharCount>1663</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities</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>44</volume><issue>5</issue><pages><first>1595</first><last>1606</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-6GP69DPW-0</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>2005</publicationDate><copyrightDate>2005</copyrightDate><doi><json:string>10.1021/bi0486740</json:string></doi><id>C37288E46CEF7E2844C1EA2F65824A1A2B4DC31E</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-6GP69DPW-0/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-6GP69DPW-0/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-6GP69DPW-0/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-6GP69DPW-0/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor
Combinations on HIV-1 Reverse-Transcriptase Activities</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2005 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2005</date><date type="Copyright" when="2005">2005</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">Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor
Combinations on HIV-1 Reverse-Transcriptase Activities</title><author xml:id="author-0000"><persName><surname>Shaw-Reid</surname><forename type="first">Cathryn A.</forename></persName><note place="foot" n="bi0486740AF3"><ref>‡</ref><p>
Department of Antiviral Research, Merck Research Laboratories.
</p></note><note place="foot" n="bi0486740AF4"><ref>§</ref><p>
Present address: Amgen, Inc., One Amgen Center Drive, Thousand
Oaks, CA 91320-1799.</p></note></author><author xml:id="author-0001"><persName><surname>Feuston</surname><forename type="first">Bradley</forename></persName><affiliation><orgName type="laboratory">Department of Antiviral Research</orgName><orgName type="laboratory">Department of Molecular Systems</orgName><orgName type="division">Department of Cardiovascular Diseases</orgName><orgName type="laboratory">Merck Research Laboratories</orgName><address><addrLine>West Point</addrLine><addrLine>Pennsylvania 19486-0004</addrLine><addrLine>and Department of MedicineDivision of Infectious Diseases</addrLine><addrLine>University of Pittsburgh</addrLine><addrLine>Pittsburgh</addrLine><addrLine>Pennsylvania 15261</addrLine></address></affiliation><note place="foot" n="bi0486740AF5"><ref>‖</ref><p>
Department of Molecular Systems, Merck Research Laboratories.
</p></note></author><author xml:id="author-0002"><persName><surname>Munshi</surname><forename type="first">Vandna</forename></persName><affiliation><orgName type="laboratory">Department of Antiviral Research</orgName><orgName type="laboratory">Department of Molecular Systems</orgName><orgName type="division">Department of Cardiovascular Diseases</orgName><orgName type="laboratory">Merck Research Laboratories</orgName><address><addrLine>West Point</addrLine><addrLine>Pennsylvania 19486-0004</addrLine><addrLine>and Department of MedicineDivision of Infectious Diseases</addrLine><addrLine>University of Pittsburgh</addrLine><addrLine>Pittsburgh</addrLine><addrLine>Pennsylvania 15261</addrLine></address></affiliation><note place="foot" n="bi0486740AF3"><ref>‡</ref><p>
Department of Antiviral Research, Merck Research Laboratories.
</p></note></author><author xml:id="author-0003"><persName><surname>Getty</surname><forename type="first">Krista</forename></persName><affiliation><orgName type="laboratory">Department of Antiviral Research</orgName><orgName type="laboratory">Department of Molecular Systems</orgName><orgName type="division">Department of Cardiovascular Diseases</orgName><orgName type="laboratory">Merck Research Laboratories</orgName><address><addrLine>West Point</addrLine><addrLine>Pennsylvania 19486-0004</addrLine><addrLine>and Department of MedicineDivision of Infectious Diseases</addrLine><addrLine>University of Pittsburgh</addrLine><addrLine>Pittsburgh</addrLine><addrLine>Pennsylvania 15261</addrLine></address></affiliation><note place="foot" n="bi0486740AF3"><ref>‡</ref><p>
Department of Antiviral Research, Merck Research Laboratories.
</p></note></author><author xml:id="author-0004"><persName><surname>Krueger</surname><forename type="first">Julie</forename></persName><affiliation><orgName type="laboratory">Department of Antiviral Research</orgName><orgName type="laboratory">Department of Molecular Systems</orgName><orgName type="division">Department of Cardiovascular Diseases</orgName><orgName type="laboratory">Merck Research Laboratories</orgName><address><addrLine>West Point</addrLine><addrLine>Pennsylvania 19486-0004</addrLine><addrLine>and Department of MedicineDivision of Infectious Diseases</addrLine><addrLine>University of Pittsburgh</addrLine><addrLine>Pittsburgh</addrLine><addrLine>Pennsylvania 15261</addrLine></address></affiliation><note place="foot" n="bi0486740AF6"><ref>⊥</ref><p>
Department of Cardiovascular Diseases, Merck Research Laboratories.</p></note></author><author xml:id="author-0005"><persName><surname>Hazuda</surname><forename type="first">Daria J.</forename></persName><affiliation><orgName type="laboratory">Department of Antiviral Research</orgName><orgName type="laboratory">Department of Molecular Systems</orgName><orgName type="division">Department of Cardiovascular Diseases</orgName><orgName type="laboratory">Merck Research Laboratories</orgName><address><addrLine>West Point</addrLine><addrLine>Pennsylvania 19486-0004</addrLine><addrLine>and Department of MedicineDivision of Infectious Diseases</addrLine><addrLine>University of Pittsburgh</addrLine><addrLine>Pittsburgh</addrLine><addrLine>Pennsylvania 15261</addrLine></address></affiliation><note place="foot" n="bi0486740AF3"><ref>‡</ref><p>
Department of Antiviral Research, Merck Research Laboratories.
</p></note></author><author xml:id="author-0006"><persName><surname>Parniak</surname><forename type="first">Michael A.</forename></persName><affiliation><orgName type="laboratory">Department of Antiviral Research</orgName><orgName type="laboratory">Department of Molecular Systems</orgName><orgName type="division">Department of Cardiovascular Diseases</orgName><orgName type="laboratory">Merck Research Laboratories</orgName><address><addrLine>West Point</addrLine><addrLine>Pennsylvania 19486-0004</addrLine><addrLine>and Department of MedicineDivision of Infectious Diseases</addrLine><addrLine>University of Pittsburgh</addrLine><addrLine>Pittsburgh</addrLine><addrLine>Pennsylvania 15261</addrLine></address></affiliation><note place="foot" n="bi0486740AF10"><ref>#</ref><p>
Department of Medicine/Division of Infectious Diseases, University
of Pittsburgh.</p></note></author><author xml:id="author-0007" role="corresp"><persName><surname>Miller</surname><forename type="first">Michael D.</forename></persName><note place="foot" n="bi0486740AF3"><ref>‡</ref><p>
Department of Antiviral Research, Merck Research Laboratories.
</p></note><affiliation role="corresp"> To whom correspondence should be addressed: Department of Antiviral Research, Merck and Co., Inc., P.O. Box 4, WP42-209, West Point, PA 19486-0004. Telephone: 215-652-0480. Fax: 215-993-5798. E-mail: michael_miller1@merck.com.</affiliation></author><author xml:id="author-0008"><persName><surname>Lewis</surname><forename type="first">Dale</forename></persName><affiliation><orgName type="laboratory">Department of Antiviral Research</orgName><orgName type="laboratory">Department of Molecular Systems</orgName><orgName type="division">Department of Cardiovascular Diseases</orgName><orgName type="laboratory">Merck Research Laboratories</orgName><address><addrLine>West Point</addrLine><addrLine>Pennsylvania 19486-0004</addrLine><addrLine>and Department of MedicineDivision of Infectious Diseases</addrLine><addrLine>University of Pittsburgh</addrLine><addrLine>Pittsburgh</addrLine><addrLine>Pennsylvania 15261</addrLine></address></affiliation><note place="foot" n="bi0486740AF6"><ref>⊥</ref><p>
Department of Cardiovascular Diseases, Merck Research Laboratories.</p></note></author><idno type="istex">C37288E46CEF7E2844C1EA2F65824A1A2B4DC31E</idno><idno type="ark">ark:/67375/TPS-6GP69DPW-0</idno><idno type="DOI">10.1021/bi0486740</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">2005</date><date type="published">2005</date><biblScope unit="vol">44</biblScope><biblScope unit="issue">5</biblScope><biblScope unit="page" from="1595">1595</biblScope><biblScope unit="page" to="1606">1606</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><graphic url="bi0486740n00001.tif"></graphic></p><p>Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H)
activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase
domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the
impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity <hi rend="italic">in vitro</hi>. The
nucleoside analogue 3‘-azido-3‘-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the
pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent
manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products
differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different
RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase
H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting
that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H
active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid
(DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H
cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although
the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA
combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens
incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.
</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-2.0.3"><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/bi0486740</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor
Combinations on HIV-1 Reverse-Transcriptase Activities</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Shaw-Reid</surname><given-names>Cathryn A.</given-names></name><xref rid="bi0486740AF3">‡</xref><xref rid="bi0486740AF4">§</xref></contrib><contrib contrib-type="author"><name><surname>Feuston</surname><given-names>Bradley</given-names></name><xref rid="bi0486740AF5">‖</xref></contrib><contrib contrib-type="author"><name><surname>Munshi</surname><given-names>Vandna</given-names></name><xref rid="bi0486740AF3">‡</xref></contrib><contrib contrib-type="author"><name><surname>Getty</surname><given-names>Krista</given-names></name><xref rid="bi0486740AF3">‡</xref></contrib><contrib contrib-type="author"><name><surname>Krueger</surname><given-names>Julie</given-names></name><xref rid="bi0486740AF6">⊥</xref></contrib><contrib contrib-type="author"><name><surname>Hazuda</surname><given-names>Daria J.</given-names></name><xref rid="bi0486740AF3">‡</xref></contrib><contrib contrib-type="author"><name><surname>Parniak</surname><given-names>Michael A.</given-names></name><xref rid="bi0486740AF10">#</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Miller</surname><given-names>Michael D.</given-names></name><xref rid="bi0486740AF1">*</xref><xref rid="bi0486740AF3">‡</xref></contrib><contrib contrib-type="author"><name><surname>Lewis</surname><given-names>Dale</given-names></name><xref rid="bi0486740AF6">⊥</xref></contrib><aff>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck Research
Laboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261
</aff></contrib-group><author-notes><fn id="bi0486740AF3"><label>‡</label><p>
Department of Antiviral Research, Merck Research Laboratories.
</p></fn><fn id="bi0486740AF4"><label>§</label><p>
Present address: Amgen, Inc., One Amgen Center Drive, Thousand
Oaks, CA 91320-1799.</p></fn><fn id="bi0486740AF5"><label>‖</label><p>
Department of Molecular Systems, Merck Research Laboratories.
</p></fn><fn id="bi0486740AF6"><label>⊥</label><p>
Department of Cardiovascular Diseases, Merck Research Laboratories.</p></fn><fn id="bi0486740AF10"><label>#</label><p>
Department of Medicine/Division of Infectious Diseases, University
of Pittsburgh.</p></fn><corresp id="bi0486740AF1">
To whom correspondence should be addressed: Department of
Antiviral Research, Merck and Co., Inc., P.O. Box 4, WP42-209, West
Point, PA 19486-0004. Telephone: 215-652-0480. Fax: 215-993-5798.
E-mail: michael_miller1@merck.com.</corresp></author-notes><pub-date pub-type="epub"><day>12</day><month>01</month><year>2005</year></pub-date><pub-date pub-type="ppub"><day>08</day><month>02</month><year>2005</year></pub-date><volume>44</volume><issue>5</issue><fpage>1595</fpage><lpage>1606</lpage><supplementary-material xlink:href="bi0486740si20041024_093717.pdf"></supplementary-material><history><date date-type="received"><day>24</day><month>06</month><year>2004</year></date><date date-type="rev-recd"><day>24</day><month>10</month><year>2004</year></date><date date-type="asap"><day>12</day><month>01</month><year>2005</year></date><date date-type="issue-pub"><day>08</day><month>02</month><year>2005</year></date></history><permissions><copyright-statement>Copyright © 2005 American Chemical Society</copyright-statement><copyright-year>2005</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><graphic content-type="abstract-graphic" xlink:href="bi0486740n00001.tif"></graphic><p>Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H)
activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase
domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the
impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity <italic>in vitro</italic>. The
nucleoside analogue 3‘-azido-3‘-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the
pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent
manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products
differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different
RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase
H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting
that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H
active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid
(DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H
cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although
the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA
combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens
incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi0486740</meta-value></custom-meta></custom-meta-group></article-meta></front><body></body><back></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities</title></titleInfo><titleInfo contentType="CDATA"><title>Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities</title></titleInfo><name type="personal"><namePart type="family">SHAW-REID</namePart><namePart type="given">Cathryn A.</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Antiviral Research, Merck Research Laboratories.</affiliation><affiliation> Present address: Amgen, Inc., One Amgen Center Drive, ThousandOaks, CA 91320-1799.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">FEUSTON</namePart><namePart type="given">Bradley</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Molecular Systems, Merck Research Laboratories.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">MUNSHI</namePart><namePart type="given">Vandna</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Antiviral Research, Merck Research Laboratories.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">GETTY</namePart><namePart type="given">Krista</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Antiviral Research, Merck Research Laboratories.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">KRUEGER</namePart><namePart type="given">Julie</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Cardiovascular Diseases, Merck Research Laboratories.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">HAZUDA</namePart><namePart type="given">Daria J.</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Antiviral Research, Merck Research Laboratories.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">PARNIAK</namePart><namePart type="given">Michael A.</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Medicine/Division of Infectious Diseases, Universityof Pittsburgh.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">MILLER</namePart><namePart type="given">Michael D.</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Antiviral Research, Merck Research Laboratories.</affiliation><affiliation> To whom correspondence should be addressed: Department ofAntiviral Research, Merck and Co., Inc., P.O. Box 4, WP42-209, WestPoint, PA 19486-0004. Telephone: 215-652-0480. Fax: 215-993-5798.E-mail: michael_miller1@merck.com.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">LEWIS</namePart><namePart type="given">Dale</namePart><affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</affiliation><affiliation> Department of Cardiovascular Diseases, Merck Research Laboratories.</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">2005-01-12</dateCreated><dateIssued encoding="w3cdtf">2005-02-08</dateIssued><copyrightDate encoding="w3cdtf">2005</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3‘-azido-3‘-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.</abstract><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>2005</date><detail type="volume"><caption>vol.</caption><number>44</number></detail><detail type="issue"><caption>no.</caption><number>5</number></detail><extent unit="pages"><start>1595</start><end>1606</end></extent></part></relatedItem><identifier type="istex">C37288E46CEF7E2844C1EA2F65824A1A2B4DC31E</identifier><identifier type="ark">ark:/67375/TPS-6GP69DPW-0</identifier><identifier type="DOI">10.1021/bi0486740</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2005 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-6GP69DPW-0/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/C37288E46CEF7E2844C1EA2F65824A1A2B4DC31E/annexes/tiff</uri></json:item><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-6GP69DPW-0/annexes.pdf</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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