Replication-Competent Chimeric Hepatitis C Virus Subgenomic Replicons
Objective: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural...
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Veröffentlicht in: | Intervirology 2005-01, Vol.48 (2-3), p.183-191 |
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creator | Lemm, Julie A. Liu, Mengping Rose, Ronald E. Fridell, Robert O’Boyle II, Donald R. Colonno, Richard Gao, Min |
description | Objective: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural genes of a 1b replicon with genotype 1a sequences. Results: A chimeric replicon containing a genotype 1a NS5A protein did not replicate in a transient assay, but could be used to establish stable cell lines using G418 selection. The cell lines contained a K1846T mutation in NS4B which functioned as an adaptive mutation that now allowed the chimera to replicate at levels similar to wild-type replicons. Similarly, replication of a 1a NS5A5B chimera was only observed after establishment of stable cell lines, even in the presence of the K1846T mutation. Sequence analysis of this cell line revealed an additional adaptive mutation of M1496L in NS3. Lastly, by including the K1846T mutation in a replicon that was entirely genotype 1a sequence, stable 1a cell lines could be established. Conclusion: These studies identify an NS4B adaptive mutation, K1846T, which allows establishment of a replication-competent 1a replicon and demonstrate the utility of this chimeric approach for establishing replicons for various HCV genotypes. |
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Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural genes of a 1b replicon with genotype 1a sequences. Results: A chimeric replicon containing a genotype 1a NS5A protein did not replicate in a transient assay, but could be used to establish stable cell lines using G418 selection. The cell lines contained a K1846T mutation in NS4B which functioned as an adaptive mutation that now allowed the chimera to replicate at levels similar to wild-type replicons. Similarly, replication of a 1a NS5A5B chimera was only observed after establishment of stable cell lines, even in the presence of the K1846T mutation. Sequence analysis of this cell line revealed an additional adaptive mutation of M1496L in NS3. Lastly, by including the K1846T mutation in a replicon that was entirely genotype 1a sequence, stable 1a cell lines could be established. Conclusion: These studies identify an NS4B adaptive mutation, K1846T, which allows establishment of a replication-competent 1a replicon and demonstrate the utility of this chimeric approach for establishing replicons for various HCV genotypes.</description><identifier>ISSN: 0300-5526</identifier><identifier>EISSN: 1423-0100</identifier><identifier>DOI: 10.1159/000081747</identifier><identifier>PMID: 15812193</identifier><identifier>CODEN: IVRYAK</identifier><language>eng</language><publisher>Basel, Switzerland: S. Karger AG</publisher><subject>Amino Acid Substitution ; Genes, Viral ; Genome, Viral ; Genotype ; Hepacivirus - genetics ; Hepacivirus - physiology ; Hepatitis C ; Hepatitis C virus ; Mutation ; Original Paper ; Replicon ; Viral Nonstructural Proteins - genetics ; Viral Nonstructural Proteins - physiology ; Virus Replication</subject><ispartof>Intervirology, 2005-01, Vol.48 (2-3), p.183-191</ispartof><rights>2005 S. Karger AG, Basel</rights><rights>Copyright (c) 2005 S. Karger AG, Basel.</rights><rights>Copyright (c) 2005 S. Karger AG, Basel</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c419t-82a92e43309080a828f91a69903b34fabb84f73dba4b77c8f0ef07f24921ea653</citedby><cites>FETCH-LOGICAL-c419t-82a92e43309080a828f91a69903b34fabb84f73dba4b77c8f0ef07f24921ea653</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,2423,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15812193$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lemm, Julie A.</creatorcontrib><creatorcontrib>Liu, Mengping</creatorcontrib><creatorcontrib>Rose, Ronald E.</creatorcontrib><creatorcontrib>Fridell, Robert</creatorcontrib><creatorcontrib>O’Boyle II, Donald R.</creatorcontrib><creatorcontrib>Colonno, Richard</creatorcontrib><creatorcontrib>Gao, Min</creatorcontrib><title>Replication-Competent Chimeric Hepatitis C Virus Subgenomic Replicons</title><title>Intervirology</title><addtitle>Intervirology</addtitle><description>Objective: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural genes of a 1b replicon with genotype 1a sequences. Results: A chimeric replicon containing a genotype 1a NS5A protein did not replicate in a transient assay, but could be used to establish stable cell lines using G418 selection. The cell lines contained a K1846T mutation in NS4B which functioned as an adaptive mutation that now allowed the chimera to replicate at levels similar to wild-type replicons. Similarly, replication of a 1a NS5A5B chimera was only observed after establishment of stable cell lines, even in the presence of the K1846T mutation. Sequence analysis of this cell line revealed an additional adaptive mutation of M1496L in NS3. Lastly, by including the K1846T mutation in a replicon that was entirely genotype 1a sequence, stable 1a cell lines could be established. Conclusion: These studies identify an NS4B adaptive mutation, K1846T, which allows establishment of a replication-competent 1a replicon and demonstrate the utility of this chimeric approach for establishing replicons for various HCV genotypes.</description><subject>Amino Acid Substitution</subject><subject>Genes, Viral</subject><subject>Genome, Viral</subject><subject>Genotype</subject><subject>Hepacivirus - genetics</subject><subject>Hepacivirus - physiology</subject><subject>Hepatitis C</subject><subject>Hepatitis C virus</subject><subject>Mutation</subject><subject>Original Paper</subject><subject>Replicon</subject><subject>Viral Nonstructural Proteins - genetics</subject><subject>Viral Nonstructural Proteins - physiology</subject><subject>Virus Replication</subject><issn>0300-5526</issn><issn>1423-0100</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNqF0c9LwzAUB_AgipvTg2dByg6Ch-rLjzbNUcp0g6Gg02tJu2R29pdJe_C_N7NzggjmksP3817IewidYrjCOBDX4E6EOeN7aIgZoT5ggH00BArgBwEJB-jI2rVTFFM4RAMcRJhgQYdo8qiaIs9km9eVH9dlo1pVtV78mpfK5Jk3VY3L2tx6sfeSm856T126UlVdurCvrSt7jA60LKw62d4j9Hw7WcRTf_5wN4tv5n7GsGj9iEhBFKMUBEQgIxJpgWUoBNCUMi3TNGKa02UqWcp5FmlQGrgmTBCsZBjQEbro-zamfu-UbZMyt5kqClmpurMJAUE4D_C_EPMgBM6pg-NfcF13pnKfcM2Ym1MYbJ697FFmamuN0klj8lKajwRDstlAstuAs-fbhl1aquWP3I7cgbMevEmzUmYHvsvHf6az-8UXSJqlpp969JLT</recordid><startdate>20050101</startdate><enddate>20050101</enddate><creator>Lemm, Julie A.</creator><creator>Liu, Mengping</creator><creator>Rose, Ronald E.</creator><creator>Fridell, Robert</creator><creator>O’Boyle II, Donald R.</creator><creator>Colonno, Richard</creator><creator>Gao, Min</creator><general>S. 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genetics</topic><topic>Hepacivirus - physiology</topic><topic>Hepatitis C</topic><topic>Hepatitis C virus</topic><topic>Mutation</topic><topic>Original Paper</topic><topic>Replicon</topic><topic>Viral Nonstructural Proteins - genetics</topic><topic>Viral Nonstructural Proteins - physiology</topic><topic>Virus Replication</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lemm, Julie A.</creatorcontrib><creatorcontrib>Liu, Mengping</creatorcontrib><creatorcontrib>Rose, Ronald E.</creatorcontrib><creatorcontrib>Fridell, Robert</creatorcontrib><creatorcontrib>O’Boyle II, Donald R.</creatorcontrib><creatorcontrib>Colonno, Richard</creatorcontrib><creatorcontrib>Gao, Min</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>STEM Database</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest Central (New)</collection><collection>ProQuest One Academic (New)</collection><collection>ProQuest Health & Medical Research Collection</collection><collection>ProQuest One Academic Middle East (New)</collection><collection>ProQuest One Health & Nursing</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Applied & Life Sciences</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>SIRS Editorial</collection><jtitle>Intervirology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lemm, Julie A.</au><au>Liu, Mengping</au><au>Rose, Ronald E.</au><au>Fridell, Robert</au><au>O’Boyle II, Donald R.</au><au>Colonno, Richard</au><au>Gao, Min</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Replication-Competent Chimeric Hepatitis C Virus Subgenomic Replicons</atitle><jtitle>Intervirology</jtitle><addtitle>Intervirology</addtitle><date>2005-01-01</date><risdate>2005</risdate><volume>48</volume><issue>2-3</issue><spage>183</spage><epage>191</epage><pages>183-191</pages><issn>0300-5526</issn><eissn>1423-0100</eissn><coden>IVRYAK</coden><abstract>Objective: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural genes of a 1b replicon with genotype 1a sequences. Results: A chimeric replicon containing a genotype 1a NS5A protein did not replicate in a transient assay, but could be used to establish stable cell lines using G418 selection. The cell lines contained a K1846T mutation in NS4B which functioned as an adaptive mutation that now allowed the chimera to replicate at levels similar to wild-type replicons. Similarly, replication of a 1a NS5A5B chimera was only observed after establishment of stable cell lines, even in the presence of the K1846T mutation. Sequence analysis of this cell line revealed an additional adaptive mutation of M1496L in NS3. Lastly, by including the K1846T mutation in a replicon that was entirely genotype 1a sequence, stable 1a cell lines could be established. Conclusion: These studies identify an NS4B adaptive mutation, K1846T, which allows establishment of a replication-competent 1a replicon and demonstrate the utility of this chimeric approach for establishing replicons for various HCV genotypes.</abstract><cop>Basel, Switzerland</cop><pub>S. Karger AG</pub><pmid>15812193</pmid><doi>10.1159/000081747</doi><tpages>9</tpages></addata></record> |
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subjects | Amino Acid Substitution Genes, Viral Genome, Viral Genotype Hepacivirus - genetics Hepacivirus - physiology Hepatitis C Hepatitis C virus Mutation Original Paper Replicon Viral Nonstructural Proteins - genetics Viral Nonstructural Proteins - physiology Virus Replication |
title | Replication-Competent Chimeric Hepatitis C Virus Subgenomic Replicons |
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