Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation
The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3′-end, we examine...
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| Veröffentlicht in: | The Journal of biological chemistry 2008-12, Vol.283 (49), p.33893-33901 |
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| creator | Ferrari, Eric He, Zhiqing Palermo, Robert E. Huang, H.-C. |
| description | The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3′-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (Kappm ∼ 100–400 μm) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 103- to 104-fold, revealing a much reduced nucleotide requirement for elongation (Kappm ∼ 0.03–0.09 μm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by |
| doi_str_mv | 10.1074/jbc.M803094200 |
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Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3′-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (Kappm ∼ 100–400 μm) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 103- to 104-fold, revealing a much reduced nucleotide requirement for elongation (Kappm ∼ 0.03–0.09 μm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 μm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3′-end of the HCV(–)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M803094200</identifier><identifier>PMID: 18840605</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Adenosine Triphosphate - chemistry ; Base Sequence ; Enzyme Catalysis and Regulation ; Escherichia coli - metabolism ; Gene Expression Regulation, Viral ; Hepacivirus - enzymology ; Heparin - chemistry ; Kinetics ; Molecular Sequence Data ; Nucleotides - chemistry ; Recombinant Proteins - chemistry ; Ribonuclease T1 - chemistry ; RNA - chemistry ; RNA-Dependent RNA Polymerase - chemistry ; RNA-Dependent RNA Polymerase - genetics ; Viral Nonstructural Proteins - chemistry ; Viral Nonstructural Proteins - physiology ; Zalcitabine - chemistry</subject><ispartof>The Journal of biological chemistry, 2008-12, Vol.283 (49), p.33893-33901</ispartof><rights>2008 © 2008 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>Copyright © 2008, The American Society for Biochemistry and Molecular Biology, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c490t-a80167843c1ba19af529cd5a132c874ecdf5c8ec8243e1d0f71a658d88a89e6c3</citedby><cites>FETCH-LOGICAL-c490t-a80167843c1ba19af529cd5a132c874ecdf5c8ec8243e1d0f71a658d88a89e6c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662215/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662215/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18840605$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ferrari, Eric</creatorcontrib><creatorcontrib>He, Zhiqing</creatorcontrib><creatorcontrib>Palermo, Robert E.</creatorcontrib><creatorcontrib>Huang, H.-C.</creatorcontrib><title>Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3′-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (Kappm ∼ 100–400 μm) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 103- to 104-fold, revealing a much reduced nucleotide requirement for elongation (Kappm ∼ 0.03–0.09 μm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 μm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3′-end of the HCV(–)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.</description><subject>Adenosine Triphosphate - chemistry</subject><subject>Base Sequence</subject><subject>Enzyme Catalysis and Regulation</subject><subject>Escherichia coli - metabolism</subject><subject>Gene Expression Regulation, Viral</subject><subject>Hepacivirus - enzymology</subject><subject>Heparin - chemistry</subject><subject>Kinetics</subject><subject>Molecular Sequence Data</subject><subject>Nucleotides - chemistry</subject><subject>Recombinant Proteins - chemistry</subject><subject>Ribonuclease T1 - chemistry</subject><subject>RNA - chemistry</subject><subject>RNA-Dependent RNA Polymerase - chemistry</subject><subject>RNA-Dependent RNA Polymerase - genetics</subject><subject>Viral Nonstructural Proteins - chemistry</subject><subject>Viral Nonstructural Proteins - physiology</subject><subject>Zalcitabine - chemistry</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kM9vFCEYQInR2LV69agcvM7Kz1m4mNh1a5vUaqw13ggD3-zQzAwrzLb2vxedxupBLoTweHx5CD2nZEnJSry-atzygyKcaMEIeYAWlChecUm_PUQLQhitNJPqAD3J-YqUJTR9jA6oUoLURC5QOIGdncIUMl7jryHtMz6_kEf4U-xvB0g2A9786EITpozfhTyF0U34fO96iFPwgD_D931IMMBYgDYmfDoWWTHGEdvR400fx-3v41P0qLV9hmd3-yG6PN58WZ9UZx_fn67fnlVOaDJVVhFar5TgjjaWattKpp2XlnLm1EqA8610CpxiggP1pF1RW0vllbJKQ-34IXoze3f7ZgDvymTJ9maXwmDTrYk2mH9vxtCZbbw2rK4Zo7IIlrPApZhzgvbPW0rMr-imRDf30cuDF3__eI_fVS7Aqxnowra7KblME6LrYDBMcSO04VxpXrCXM9baaOw2hWwuLxihnFBZi1rTQqiZgBLwOkAy2QUYHfgidZPxMfxvyJ_aQKfm</recordid><startdate>20081205</startdate><enddate>20081205</enddate><creator>Ferrari, Eric</creator><creator>He, Zhiqing</creator><creator>Palermo, Robert E.</creator><creator>Huang, H.-C.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>5PM</scope></search><sort><creationdate>20081205</creationdate><title>Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation</title><author>Ferrari, Eric ; He, Zhiqing ; Palermo, Robert E. ; Huang, H.-C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c490t-a80167843c1ba19af529cd5a132c874ecdf5c8ec8243e1d0f71a658d88a89e6c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Adenosine Triphosphate - chemistry</topic><topic>Base Sequence</topic><topic>Enzyme Catalysis and Regulation</topic><topic>Escherichia coli - metabolism</topic><topic>Gene Expression Regulation, Viral</topic><topic>Hepacivirus - enzymology</topic><topic>Heparin - chemistry</topic><topic>Kinetics</topic><topic>Molecular Sequence Data</topic><topic>Nucleotides - chemistry</topic><topic>Recombinant Proteins - chemistry</topic><topic>Ribonuclease T1 - chemistry</topic><topic>RNA - chemistry</topic><topic>RNA-Dependent RNA Polymerase - chemistry</topic><topic>RNA-Dependent RNA Polymerase - genetics</topic><topic>Viral Nonstructural Proteins - chemistry</topic><topic>Viral Nonstructural Proteins - physiology</topic><topic>Zalcitabine - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ferrari, Eric</creatorcontrib><creatorcontrib>He, Zhiqing</creatorcontrib><creatorcontrib>Palermo, Robert E.</creatorcontrib><creatorcontrib>Huang, H.-C.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ferrari, Eric</au><au>He, Zhiqing</au><au>Palermo, Robert E.</au><au>Huang, H.-C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2008-12-05</date><risdate>2008</risdate><volume>283</volume><issue>49</issue><spage>33893</spage><epage>33901</epage><pages>33893-33901</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3′-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (Kappm ∼ 100–400 μm) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 103- to 104-fold, revealing a much reduced nucleotide requirement for elongation (Kappm ∼ 0.03–0.09 μm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 μm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3′-end of the HCV(–)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>18840605</pmid><doi>10.1074/jbc.M803094200</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Alma/SFX Local Collection |
| subjects | Adenosine Triphosphate - chemistry Base Sequence Enzyme Catalysis and Regulation Escherichia coli - metabolism Gene Expression Regulation, Viral Hepacivirus - enzymology Heparin - chemistry Kinetics Molecular Sequence Data Nucleotides - chemistry Recombinant Proteins - chemistry Ribonuclease T1 - chemistry RNA - chemistry RNA-Dependent RNA Polymerase - chemistry RNA-Dependent RNA Polymerase - genetics Viral Nonstructural Proteins - chemistry Viral Nonstructural Proteins - physiology Zalcitabine - chemistry |
| title | Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation |
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