Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid
Identifieur interne : 001F01 ( Istex/Corpus ); précédent : 001F00; suivant : 001F02Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid
Auteurs : Reet Kurg ; Ülo Langel ; Mart UstavSource :
- Virus Research [ 0168-1702 ] ; 2000.
English descriptors
- KwdEn :
- Teeft :
- Acad, Assay, Assay conditions, Binding, Binding buffer, Binding site, Binding sites, Bovine, Bovine papillomavirus, Bovine papillomavirus type, Buffer conditions, Dsdna, Dsdna target, Duplex, E2bs, Egholm, Electroporation, Free target, Hybrid, Independent experiments, Kurg, Linearizing enzyme hindiii, Master regulator, Native polyacrylamide, Natl, Nielsen, Oligonucleotide, Papillomavirus, Papillomavirus origin, Peptide, Physiological salt concentrations, Plasmid, Pna1, Pna2, Pnas, Present study, Proc, Protein activity, Protein binding, Pucalu, Recognition sequence, Replication, Replication assay, Reporter plasmid pucalu, Room temperature, Salt concentration, Stable complexes, Stenlund, Target site, Transcription, Transient replication, Ustav, Various concentrations, Viral, Virus research.
Abstract
Abstract: The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific inhibitor of the E2 protein activity is studied in this report. We demonstrate that replacement of one or both DNA strands with the complementary PNA reduced drastically the affinity of the BPV-1 E2 protein to its target site in the direct as well as in competitive binding as shown by in vitro gel-shift assays. We demonstrate that PNA could specifically bind to the double stranded E2 binding site by forming the complex with DNA oligonucleotide. In addition, PNA was able to bind specifically to the E2 binding site within the supercoiled plasmid DNA. Such binding of PNA to the E2 binding site within the origin of replication specifically abolished the activity of the E2 protein in the initiation of DNA replication in vivo.
Url:
DOI: 10.1016/S0168-1702(99)00124-0
Links to Exploration step
ISTEX:B0A035B28CDD3BF7DEC3F1535465CE77161E2F64Le document en format XML
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ustav@ebc.ee</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:B0A035B28CDD3BF7DEC3F1535465CE77161E2F64</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1016/S0168-1702(99)00124-0</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-9VKQX3LX-W/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001F01</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001F01</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid</title><author><name sortKey="Kurg, Reet" sort="Kurg, Reet" uniqKey="Kurg R" first="Reet" last="Kurg">Reet Kurg</name><affiliation><mods:affiliation>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</mods:affiliation></affiliation></author><author><name sortKey="Langel, Ulo" sort="Langel, Ulo" uniqKey="Langel U" first="Ülo" last="Langel">Ülo Langel</name><affiliation><mods:affiliation>Department of Neurochemistry and Neurotoxicology, Arrheniuslaboratories, Stockholm University, S-10691 Stockholm, Sweden</mods:affiliation></affiliation></author><author><name sortKey="Ustav, Mart" sort="Ustav, Mart" uniqKey="Ustav M" first="Mart" last="Ustav">Mart Ustav</name><affiliation><mods:affiliation>E-mail: ustav@ebc.ee</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Virus Research</title><title level="j" type="abbrev">VIRUS</title><idno type="ISSN">0168-1702</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">66</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="39">39</biblScope><biblScope unit="page" to="50">50</biblScope></imprint><idno type="ISSN">0168-1702</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0168-1702</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>E2 protein</term><term>Papillomavirus</term><term>Peptide nucleic acid (PNA)</term><term>Replication</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Acad</term><term>Assay</term><term>Assay conditions</term><term>Binding</term><term>Binding buffer</term><term>Binding site</term><term>Binding sites</term><term>Bovine</term><term>Bovine papillomavirus</term><term>Bovine papillomavirus type</term><term>Buffer conditions</term><term>Dsdna</term><term>Dsdna target</term><term>Duplex</term><term>E2bs</term><term>Egholm</term><term>Electroporation</term><term>Free target</term><term>Hybrid</term><term>Independent experiments</term><term>Kurg</term><term>Linearizing enzyme hindiii</term><term>Master regulator</term><term>Native polyacrylamide</term><term>Natl</term><term>Nielsen</term><term>Oligonucleotide</term><term>Papillomavirus</term><term>Papillomavirus origin</term><term>Peptide</term><term>Physiological salt concentrations</term><term>Plasmid</term><term>Pna1</term><term>Pna2</term><term>Pnas</term><term>Present study</term><term>Proc</term><term>Protein activity</term><term>Protein binding</term><term>Pucalu</term><term>Recognition sequence</term><term>Replication</term><term>Replication assay</term><term>Reporter plasmid pucalu</term><term>Room temperature</term><term>Salt concentration</term><term>Stable complexes</term><term>Stenlund</term><term>Target site</term><term>Transcription</term><term>Transient replication</term><term>Ustav</term><term>Various concentrations</term><term>Viral</term><term>Virus research</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific inhibitor of the E2 protein activity is studied in this report. We demonstrate that replacement of one or both DNA strands with the complementary PNA reduced drastically the affinity of the BPV-1 E2 protein to its target site in the direct as well as in competitive binding as shown by in vitro gel-shift assays. We demonstrate that PNA could specifically bind to the double stranded E2 binding site by forming the complex with DNA oligonucleotide. In addition, PNA was able to bind specifically to the E2 binding site within the supercoiled plasmid DNA. Such binding of PNA to the E2 binding site within the origin of replication specifically abolished the activity of the E2 protein in the initiation of DNA replication in vivo.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>papillomavirus</json:string><json:string>plasmid</json:string><json:string>e2bs</json:string><json:string>replication</json:string><json:string>ustav</json:string><json:string>pna1</json:string><json:string>viral</json:string><json:string>dsdna</json:string><json:string>kurg</json:string><json:string>binding site</json:string><json:string>stenlund</json:string><json:string>pucalu</json:string><json:string>virus research</json:string><json:string>duplex</json:string><json:string>nielsen</json:string><json:string>proc</json:string><json:string>natl</json:string><json:string>target site</json:string><json:string>acad</json:string><json:string>pnas</json:string><json:string>egholm</json:string><json:string>electroporation</json:string><json:string>bovine papillomavirus</json:string><json:string>peptide</json:string><json:string>assay</json:string><json:string>oligonucleotide</json:string><json:string>pna2</json:string><json:string>hybrid</json:string><json:string>binding buffer</json:string><json:string>protein binding</json:string><json:string>papillomavirus origin</json:string><json:string>various concentrations</json:string><json:string>binding sites</json:string><json:string>buffer conditions</json:string><json:string>bovine</json:string><json:string>native polyacrylamide</json:string><json:string>recognition sequence</json:string><json:string>stable complexes</json:string><json:string>room temperature</json:string><json:string>replication assay</json:string><json:string>present study</json:string><json:string>protein activity</json:string><json:string>linearizing enzyme hindiii</json:string><json:string>assay conditions</json:string><json:string>bovine papillomavirus type</json:string><json:string>independent experiments</json:string><json:string>salt concentration</json:string><json:string>physiological salt concentrations</json:string><json:string>transient replication</json:string><json:string>reporter plasmid pucalu</json:string><json:string>master regulator</json:string><json:string>free target</json:string><json:string>dsdna target</json:string><json:string>transcription</json:string><json:string>binding</json:string></teeft></keywords><author><json:item><name>Reet Kurg</name><affiliations><json:string>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</json:string></affiliations></json:item><json:item><name>Ülo Langel</name><affiliations><json:string>Department of Neurochemistry and Neurotoxicology, Arrheniuslaboratories, Stockholm University, S-10691 Stockholm, Sweden</json:string></affiliations></json:item><json:item><name>Mart Ustav</name><affiliations><json:string>E-mail: ustav@ebc.ee</json:string><json:string>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Papillomavirus</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>E2 protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Peptide nucleic acid (PNA)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Replication</value></json:item></subject><arkIstex>ark:/67375/6H6-9VKQX3LX-W</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific inhibitor of the E2 protein activity is studied in this report. We demonstrate that replacement of one or both DNA strands with the complementary PNA reduced drastically the affinity of the BPV-1 E2 protein to its target site in the direct as well as in competitive binding as shown by in vitro gel-shift assays. We demonstrate that PNA could specifically bind to the double stranded E2 binding site by forming the complex with DNA oligonucleotide. In addition, PNA was able to bind specifically to the E2 binding site within the supercoiled plasmid DNA. Such binding of PNA to the E2 binding site within the origin of replication specifically abolished the activity of the E2 protein in the initiation of DNA replication in vivo.</abstract><qualityIndicators><score>9.208</score><pdfWordCount>6074</pdfWordCount><pdfCharCount>35910</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>12</pdfPageCount><pdfPageSize>536 x 674 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>184</abstractWordCount><abstractCharCount>1151</abstractCharCount><keywordCount>4</keywordCount></qualityIndicators><title>Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid</title><pmid><json:string>10653916</json:string></pmid><pii><json:string>S0168-1702(99)00124-0</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Virus Research</title><language><json:string>unknown</json:string></language><publicationDate>2000</publicationDate><issn><json:string>0168-1702</json:string></issn><pii><json:string>S0168-1702(00)X0060-3</json:string></pii><volume>66</volume><issue>1</issue><pages><first>39</first><last>50</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2000</json:string></date><geogName></geogName><orgName><json:string>Institute of Molecular and Cell Biology</json:string><json:string>Sweden Received</json:string><json:string>Amersham</json:string><json:string>Department of Neurochemistry</json:string><json:string>Estonian Science Foundation</json:string><json:string>Swedish Research Council for Natural Sciences</json:string><json:string>NFR</json:string></orgName><orgName_funder><json:string>Estonian Science Foundation</json:string><json:string>Swedish Research Council for Natural Sciences</json:string><json:string>NFR</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Aare</json:string><json:string>Ustav</json:string><json:string>Stenlund</json:string><json:string>Margus Pooga</json:string></persName><placeName><json:string>Mannheim</json:string><json:string>Stockholm</json:string><json:string>Tartu</json:string><json:string>Estonia</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Mohr et al., 1990</json:string><json:string>Egholm et al., 1993</json:string><json:string>Armitage et al., 1998</json:string><json:string>Langel et al., 1992</json:string><json:string>Demczuk et al., 1993</json:string><json:string>Cherny et al., 1993</json:string><json:string>Nielsen et al., 1993</json:string><json:string>Rozenberg et al., 1998</json:string><json:string>Yang et al., 1991</json:string><json:string>Peffer et al., 1993</json:string><json:string>Sedman and Stenlund, 1995</json:string><json:string>Wittung et al., 1997</json:string><json:string>Piirsoo et al., 1996</json:string><json:string>Giri and Yaniv, 1988</json:string><json:string>Nielsen et al. 1994</json:string><json:string>Hajduk et al., 1997</json:string><json:string>Gillette and Borowiec, 1998</json:string><json:string>Demidov et al., 1995</json:string><json:string>Ustav and Stenlund, 1991</json:string><json:string>Nielsen et al., 1991</json:string><json:string>GambacortiPasserini et al., 1996</json:string><json:string>Hines et al. 1998</json:string><json:string>Androphy et al., 1987</json:string><json:string>Hanvey et al., 1992</json:string><json:string>Lehman and Botchan, 1998</json:string><json:string>Eriksson and Nielsen, 1996</json:string><json:string>Demidov et al., 1994</json:string><json:string>Lim et al., 1998</json:string><json:string>for review Ustav and Ustav, 1998</json:string><json:string>Lusky et al., 1994</json:string><json:string>Ustav et al., 1991, 1993</json:string><json:string>Seo et al., 1993</json:string><json:string>R. Kurg et al.</json:string><json:string>Hegde et al., 1992</json:string><json:string>Ustav et al., 1993</json:string><json:string>Kurg et al., 1999</json:string><json:string>Vickers et al., 1995</json:string><json:string>Skiadopoulus and McBride, 1998</json:string><json:string>Ilves et al., 1999</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-9VKQX3LX-W</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - virology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - virology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Cancer Research</json:string><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Infectious Diseases</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Immunology and Microbiology</json:string><json:string>3 - Virology</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 biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1016/S0168-1702(99)00124-0</json:string></doi><id>B0A035B28CDD3BF7DEC3F1535465CE77161E2F64</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-9VKQX3LX-W/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-9VKQX3LX-W/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-9VKQX3LX-W/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©2000 Elsevier Science B.V.</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>2000</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Fig. 1: Schematic representation of E2 binding site, PNAs and papillomavirus Ori constructs used in this study. (A) Oligonucleotide and PNA sequences. The E2 binding site is shown in block letters. The PNA-s are oriented from the N to the C terminus (the C terminus is a carboxy-amide) such that when the N-terminal end of the PNA faces the 5′ end of the oligonucleotide the complex is termed parallel. (B) Schematic representation of the BPV-1 replication origin inserts used. Numbers indicate positions on the BPV-1 nucleotide sequence.</note><note type="content">Fig. 2: The ability of the BPV-1 E2 protein to bind PNA-DNA hybrid and PNA–PNA duplex. (A) The mobility shift assay was carried out with 1 ng of E2, 1 nM of end-labelled E2BS and various concentrations of pre-formed, non-labelled dsDNA, PNA–DNA hybrid or PNA–PNA complex for 1 h at room temperature. Protein–DNA complex was resolved on 6% PAGE in 0.25× Tris-borate-EDTA. (B) Quantitation of data from Fig. 2A. The values shown are the results of three independent experiments.</note><note type="content">Fig. 3: The ability of PNA-s to bind E2 binding site. (A) A fixed concentration of the end-labelled duplex E2BS (1.4 nM) was incubated with increasing concentrations of each PNA in TE for 10 min at 70°C and after that for 30 min at 37°C. The samples were analysed by electrophoresis in a 8% polyacrylamide gel, followed by autoradiography. PNA binding generates a complex of reduced mobility relative to the duplex. (B) Quantitative analysis of Fig. 3A. (C) The ability of PNA-s to bind E2BS for 2 h at 37°C. The values shown are the results of three independent experiments.</note><note type="content">Fig. 4: Effect of PNA-s on the E2 protein binding in vitro. E2BS was pre-incubated in TE buffer with PNA at the indicated concentrations for 2 h at 37°C. Following the pre-incubation, buffer conditions were adjusted to E2 binding buffer and 1 ng of the purified E2 protein was added. After 15 min incubation, E2–DNA complex and PNA–DNA complex were separated from free target by electrophoresis through a 6% native polyacrylamide gel.</note><note type="content">Fig. 5: Effect of PNA on BPV-1 origin replication. (A) Detection of the PNA and DNA components of the PNA–plasmid complex. Biotinylated-PNA1 (12 μg) was incubated with 100 ng of various papillomavirus replication origins in TE at 37°C overnight. The resulting complexes were separated in a standard agarose gel-electrophoresis (lanes 1–5) and then transferred by electroblotting. (Lanes 6–7) Immunodetection of biotinylated-PNA on PVDF membrane. (B) Effect of PNA-s on different BPV-1 origin replication. Reporter plasmids were pre-incubated with indicated concentrations of PNA in TE buffer overnight at 37°C. The plasmid-PNA complexes were then electroporated into CHO cells as described in Section 2. Episomal DNA was extracted from cells 48 h after transfection, digested with DpnI and linearizing enzyme HindIII and analyzed by Southern blotting. The replication signals of three independent experiments were quantified with a Phosphoimager, and signals from cells transfected with the origin-containing plasmids only were used as a control to normalize the results. (C) Southern blot analysis of transient replication of the BPV-1 origin plasmid pUCAlu in the presence of various concentrations of PNA. Reporter plasmid pUCAlu was pre-incubated with indicated concentrations of PNA in TE buffer overnight at 37°C. The plasmid–PNA complex was then electroporated into CHO cells, episomal DNA was extracted from cells either 48 or 72 h after transfection. Filters were probed with radiolabelled pUCAlu plasmid. M- marker, 100 pg of reporter plasmid pUCAlu. (D) Southern blot analysis of transient replication of the BPV-1 origin plasmid pUCAlu in the presence of 12 μg of PNA. In this case, DNA and PNA were mixed before co-transfection into CHO cells.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid</title><author xml:id="author-0000"><persName><forename type="first">Reet</forename><surname>Kurg</surname></persName><affiliation>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Ülo</forename><surname>Langel</surname></persName><affiliation>Department of Neurochemistry and Neurotoxicology, Arrheniuslaboratories, Stockholm University, S-10691 Stockholm, Sweden</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Mart</forename><surname>Ustav</surname></persName><email>ustav@ebc.ee</email><note type="correspondence"><p>Corresponding author. Tel.: +372-7-375047; fax: +372-7-420286</p></note><affiliation>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</affiliation></author><idno type="istex">B0A035B28CDD3BF7DEC3F1535465CE77161E2F64</idno><idno type="ark">ark:/67375/6H6-9VKQX3LX-W</idno><idno type="DOI">10.1016/S0168-1702(99)00124-0</idno><idno type="PII">S0168-1702(99)00124-0</idno></analytic><monogr><title level="j">Virus Research</title><title level="j" type="abbrev">VIRUS</title><idno type="pISSN">0168-1702</idno><idno type="PII">S0168-1702(00)X0060-3</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">66</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="39">39</biblScope><biblScope unit="page" to="50">50</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2000</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific inhibitor of the E2 protein activity is studied in this report. We demonstrate that replacement of one or both DNA strands with the complementary PNA reduced drastically the affinity of the BPV-1 E2 protein to its target site in the direct as well as in competitive binding as shown by in vitro gel-shift assays. We demonstrate that PNA could specifically bind to the double stranded E2 binding site by forming the complex with DNA oligonucleotide. In addition, PNA was able to bind specifically to the E2 binding site within the supercoiled plasmid DNA. Such binding of PNA to the E2 binding site within the origin of replication specifically abolished the activity of the E2 protein in the initiation of DNA replication in vivo.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Papillomavirus</term></item><item><term>E2 protein</term></item><item><term>Peptide nucleic acid (PNA)</term></item><item><term>Replication</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1999-10-04">Modified</change><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-9VKQX3LX-W/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2a" NDATA="IMAGE" name="gr2a"></istex:entity><istex:entity SYSTEM="gr2b" NDATA="IMAGE" name="gr2b"></istex:entity><istex:entity SYSTEM="gr3a" NDATA="IMAGE" name="gr3a"></istex:entity><istex:entity SYSTEM="gr3bc" NDATA="IMAGE" name="gr3bc"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5a" NDATA="IMAGE" name="gr5a"></istex:entity><istex:entity SYSTEM="gr5c" NDATA="IMAGE" name="gr5c"></istex:entity><istex:entity SYSTEM="gr5d" NDATA="IMAGE" name="gr5d"></istex:entity><istex:entity SYSTEM="gr5b" NDATA="IMAGE" name="gr5b"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>VIRUS</jid><aid>1821</aid><ce:pii>S0168-1702(99)00124-0</ce:pii><ce:doi>10.1016/S0168-1702(99)00124-0</ce:doi><ce:copyright type="full-transfer" year="2000">Elsevier Science B.V.</ce:copyright></item-info><head><ce:title>Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid</ce:title><ce:author-group><ce:author><ce:given-name>Reet</ce:given-name><ce:surname>Kurg</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Ülo</ce:given-name><ce:surname>Langel</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Mart</ce:given-name><ce:surname>Ustav</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref><ce:e-address>ustav@ebc.ee</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>Department of Neurochemistry and Neurotoxicology, Arrheniuslaboratories, Stockholm University, S-10691 Stockholm, Sweden</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +372-7-375047; fax: +372-7-420286</ce:text></ce:correspondence></ce:author-group><ce:date-received day="28" month="6" year="1999"></ce:date-received><ce:date-revised day="4" month="10" year="1999"></ce:date-revised><ce:date-accepted day="4" month="10" year="1999"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific inhibitor of the E2 protein activity is studied in this report. We demonstrate that replacement of one or both DNA strands with the complementary PNA reduced drastically the affinity of the BPV-1 E2 protein to its target site in the direct as well as in competitive binding as shown by in vitro gel-shift assays. We demonstrate that PNA could specifically bind to the double stranded E2 binding site by forming the complex with DNA oligonucleotide. In addition, PNA was able to bind specifically to the E2 binding site within the supercoiled plasmid DNA. Such binding of PNA to the E2 binding site within the origin of replication specifically abolished the activity of the E2 protein in the initiation of DNA replication in vivo.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Papillomavirus</ce:text></ce:keyword><ce:keyword><ce:text>E2 protein</ce:text></ce:keyword><ce:keyword><ce:text>Peptide nucleic acid (PNA)</ce:text></ce:keyword><ce:keyword><ce:text>Replication</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid</title></titleInfo><name type="personal"><namePart type="given">Reet</namePart><namePart type="family">Kurg</namePart><affiliation>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Ülo</namePart><namePart type="family">Langel</namePart><affiliation>Department of Neurochemistry and Neurotoxicology, Arrheniuslaboratories, Stockholm University, S-10691 Stockholm, Sweden</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Mart</namePart><namePart type="family">Ustav</namePart><affiliation>E-mail: ustav@ebc.ee</affiliation><affiliation>Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia</affiliation><description>Corresponding author. Tel.: +372-7-375047; fax: +372-7-420286</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length 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>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><dateModified encoding="w3cdtf">1999-10-04</dateModified><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific inhibitor of the E2 protein activity is studied in this report. We demonstrate that replacement of one or both DNA strands with the complementary PNA reduced drastically the affinity of the BPV-1 E2 protein to its target site in the direct as well as in competitive binding as shown by in vitro gel-shift assays. We demonstrate that PNA could specifically bind to the double stranded E2 binding site by forming the complex with DNA oligonucleotide. In addition, PNA was able to bind specifically to the E2 binding site within the supercoiled plasmid DNA. Such binding of PNA to the E2 binding site within the origin of replication specifically abolished the activity of the E2 protein in the initiation of DNA replication in vivo.</abstract><note type="content">Fig. 1: Schematic representation of E2 binding site, PNAs and papillomavirus Ori constructs used in this study. (A) Oligonucleotide and PNA sequences. The E2 binding site is shown in block letters. The PNA-s are oriented from the N to the C terminus (the C terminus is a carboxy-amide) such that when the N-terminal end of the PNA faces the 5′ end of the oligonucleotide the complex is termed parallel. (B) Schematic representation of the BPV-1 replication origin inserts used. Numbers indicate positions on the BPV-1 nucleotide sequence.</note><note type="content">Fig. 2: The ability of the BPV-1 E2 protein to bind PNA-DNA hybrid and PNA–PNA duplex. (A) The mobility shift assay was carried out with 1 ng of E2, 1 nM of end-labelled E2BS and various concentrations of pre-formed, non-labelled dsDNA, PNA–DNA hybrid or PNA–PNA complex for 1 h at room temperature. Protein–DNA complex was resolved on 6% PAGE in 0.25× Tris-borate-EDTA. (B) Quantitation of data from Fig. 2A. The values shown are the results of three independent experiments.</note><note type="content">Fig. 3: The ability of PNA-s to bind E2 binding site. (A) A fixed concentration of the end-labelled duplex E2BS (1.4 nM) was incubated with increasing concentrations of each PNA in TE for 10 min at 70°C and after that for 30 min at 37°C. The samples were analysed by electrophoresis in a 8% polyacrylamide gel, followed by autoradiography. PNA binding generates a complex of reduced mobility relative to the duplex. (B) Quantitative analysis of Fig. 3A. (C) The ability of PNA-s to bind E2BS for 2 h at 37°C. The values shown are the results of three independent experiments.</note><note type="content">Fig. 4: Effect of PNA-s on the E2 protein binding in vitro. E2BS was pre-incubated in TE buffer with PNA at the indicated concentrations for 2 h at 37°C. Following the pre-incubation, buffer conditions were adjusted to E2 binding buffer and 1 ng of the purified E2 protein was added. After 15 min incubation, E2–DNA complex and PNA–DNA complex were separated from free target by electrophoresis through a 6% native polyacrylamide gel.</note><note type="content">Fig. 5: Effect of PNA on BPV-1 origin replication. (A) Detection of the PNA and DNA components of the PNA–plasmid complex. Biotinylated-PNA1 (12 μg) was incubated with 100 ng of various papillomavirus replication origins in TE at 37°C overnight. The resulting complexes were separated in a standard agarose gel-electrophoresis (lanes 1–5) and then transferred by electroblotting. (Lanes 6–7) Immunodetection of biotinylated-PNA on PVDF membrane. (B) Effect of PNA-s on different BPV-1 origin replication. Reporter plasmids were pre-incubated with indicated concentrations of PNA in TE buffer overnight at 37°C. The plasmid-PNA complexes were then electroporated into CHO cells as described in Section 2. Episomal DNA was extracted from cells 48 h after transfection, digested with DpnI and linearizing enzyme HindIII and analyzed by Southern blotting. The replication signals of three independent experiments were quantified with a Phosphoimager, and signals from cells transfected with the origin-containing plasmids only were used as a control to normalize the results. (C) Southern blot analysis of transient replication of the BPV-1 origin plasmid pUCAlu in the presence of various concentrations of PNA. Reporter plasmid pUCAlu was pre-incubated with indicated concentrations of PNA in TE buffer overnight at 37°C. The plasmid–PNA complex was then electroporated into CHO cells, episomal DNA was extracted from cells either 48 or 72 h after transfection. Filters were probed with radiolabelled pUCAlu plasmid. M- marker, 100 pg of reporter plasmid pUCAlu. (D) Southern blot analysis of transient replication of the BPV-1 origin plasmid pUCAlu in the presence of 12 μg of PNA. In this case, DNA and PNA were mixed before co-transfection into CHO cells.</note><subject lang="en"><genre>Keywords</genre><topic>Papillomavirus</topic><topic>E2 protein</topic><topic>Peptide nucleic acid (PNA)</topic><topic>Replication</topic></subject><relatedItem type="host"><titleInfo><title>Virus Research</title></titleInfo><titleInfo type="abbreviated"><title>VIRUS</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><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued></originInfo><identifier type="ISSN">0168-1702</identifier><identifier type="PII">S0168-1702(00)X0060-3</identifier><part><date>2000</date><detail type="volume"><number>66</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>116</end></extent><extent unit="pages"><start>39</start><end>50</end></extent></part></relatedItem><identifier type="istex">B0A035B28CDD3BF7DEC3F1535465CE77161E2F64</identifier><identifier type="ark">ark:/67375/6H6-9VKQX3LX-W</identifier><identifier type="DOI">10.1016/S0168-1702(99)00124-0</identifier><identifier type="PII">S0168-1702(99)00124-0</identifier><accessCondition type="use and reproduction" contentType="copyright">©2000 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science B.V., ©2000</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-9VKQX3LX-W/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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